WO2017158092A1 - Anti-tnfalpha-antibodies and functional fragments thereof - Google Patents

Anti-tnfalpha-antibodies and functional fragments thereof Download PDF

Info

Publication number
WO2017158092A1
WO2017158092A1 PCT/EP2017/056237 EP2017056237W WO2017158092A1 WO 2017158092 A1 WO2017158092 A1 WO 2017158092A1 EP 2017056237 W EP2017056237 W EP 2017056237W WO 2017158092 A1 WO2017158092 A1 WO 2017158092A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
tnfa
functional fragment
seq
amino acid
Prior art date
Application number
PCT/EP2017/056237
Other languages
English (en)
French (fr)
Inventor
Tea Gunde
Sebastian Meyer
Esther Maria FURRER
Original Assignee
Tillotts Pharma Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to MX2018011258A priority Critical patent/MX2018011258A/es
Priority to CR20180448A priority patent/CR20180448A/es
Priority to RS20231087A priority patent/RS64830B1/sr
Application filed by Tillotts Pharma Ag filed Critical Tillotts Pharma Ag
Priority to BR112018016667A priority patent/BR112018016667A2/pt
Priority to EP17710748.9A priority patent/EP3430043B1/de
Priority to KR1020187022632A priority patent/KR102470235B1/ko
Priority to PL17710748.9T priority patent/PL3430043T3/pl
Priority to HRP20231464TT priority patent/HRP20231464T1/hr
Priority to MYPI2018001566A priority patent/MY194041A/en
Priority to JP2019500013A priority patent/JP7129398B2/ja
Priority to DK17710748.9T priority patent/DK3430043T5/da
Priority to CN201780015298.5A priority patent/CN109071650B/zh
Priority to FIEP17710748.9T priority patent/FI3430043T3/fi
Priority to LTEPPCT/EP2017/056237T priority patent/LT3430043T/lt
Priority to SG11201807062RA priority patent/SG11201807062RA/en
Priority to US16/085,506 priority patent/US11459383B2/en
Priority to EP23192170.1A priority patent/EP4275745A3/de
Priority to CA3011784A priority patent/CA3011784C/en
Priority to SI201731446T priority patent/SI3430043T1/sl
Priority to ES17710748T priority patent/ES2963221T3/es
Priority to KR1020227040533A priority patent/KR20220158877A/ko
Priority to MA43717A priority patent/MA43717B1/fr
Priority to MDE20190099T priority patent/MD3430043T2/ro
Priority to NZ745999A priority patent/NZ745999A/en
Priority to TNP/2018/000298A priority patent/TN2018000298A1/en
Priority to EA201891788A priority patent/EA039760B1/ru
Priority to AU2017235387A priority patent/AU2017235387B2/en
Publication of WO2017158092A1 publication Critical patent/WO2017158092A1/en
Priority to IL261031A priority patent/IL261031A/en
Priority to PH12018501907A priority patent/PH12018501907A1/en
Priority to SA518400027A priority patent/SA518400027B1/ar
Priority to CONC2018/0009783A priority patent/CO2018009783A2/es
Priority to JP2022091271A priority patent/JP2022130407A/ja
Priority to US17/890,857 priority patent/US12043663B2/en
Priority to US17/958,759 priority patent/US20230279091A1/en
Priority to AU2024219840A priority patent/AU2024219840A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to antibody molecules and functional fragments thereof, capable of binding to tumor necrosis factor alpha (TNFa), to processes for their production, and to their therapeutic uses.
  • TNFa tumor necrosis factor alpha
  • TNFa is a homo-trimeric pro-inflammatory cytokine that is released by and interacts with cells of the immune system. TNFa has also been shown to be up-regulated in a number of human diseases, including chronic diseases such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.
  • anti-TNFa antibodies in the treatment of rheumatoid arthritis and Crohn's disease is discussed in Feldman et al. (Transplantation Proceedings, 30, 4126-4127, 1998), Adorini et al. (Trends in Immunology Today, 18, 209-21 1 , 1997) and in Feldman et al. (Advances in Immunology, 64, 283-350, 1997).
  • the antibodies to TNFa previously used in such treatments are generally chimeric antibodies, such as those described in U.S. Pat. No. 5,919,452.
  • Monoclonal antibodies against TNFa have been described in the prior art. Meager et al. (Hybridoma, 6, 305-31 1 , 1987) describe murine monoclonal antibodies against recombinant TNFa. Fendly et al. (Hybridoma, 6, 359-370, 1987) describe the use of murine monoclonal antibodies against recombinant TNFa in defining neutralising epitopes on TNFa. Furthermore, in International Patent Application WO 92/1 1383, recombinant antibodies, including CDR-grafted antibodies, specific for TNFa are disclosed. Rankin et al. (British J.
  • the prior art recombinant anti-TNFa antibody molecules generally have a reduced affinity for TNFa compared to the antibodies from which the hypervariable regions or CDRs are derived. All currently marketed inhibitors of TNFa are administered intravenously or subcutaneously in weekly or longer intervals as bolus injections, resulting in high starting concentrations that are steadily decreasing until the next injection.
  • infliximab a chimeric IgG anti- human monoclonal antibody
  • etanercept a TNFR2 dimeric fusion protein, with an lgG1 Fc
  • adalimumab a fully human monoclonal antibody (mAb)
  • Humira® Kupper H et al: “Adalimumab (Humira)", Handbook of Therapeutic Antibodies, WILEY-VCH; Weinheim, 2007-01 -01 , p.697-732
  • certolizumab a PEGylated Fab fragment
  • Infliximab has a relatively low affinity to TNFa (K D > 0.2 nM; Weir et al., 2006, Therapy 3: 535) and a limited neutralization potency in an L929 assay.
  • infliximab shows substantially no cross-reactivity with TNFa from Cynomolgus or Rhesus monkeys.
  • cross-reactivity with TNFa from monkeys is highly desirable, as this allows for animal tests with primates, reflecting the situation in humans in many aspects.
  • Etanercept although a bivalent molecule, binds TNFa at a ratio of one trimer per one etanercept molecule, precluding the formation of large antigen-biotherapeutics complexes (Wallis, 2008, Lancet Infect Dis, 8: 601 ). It does not inhibit LPS-induced cytokine secretion in monocytes (Kirchner et al., 2004, Cytokine, 28: 67).
  • the potency of adalimumab is similar to that of infliximab.
  • Another disadvantage of adalimumab is its poor stability, e.g. as determined in a thermal unfolding test.
  • T m The melting temperature of adalimumab in such a test was determined to be 67.5 °C.
  • a lower T m makes antibodies less suitable for pharmaceutical use, e.g. for oral administration.
  • the potency of certolizumab is slightly greater than that of infliximab, but still not satisfying. Certolizumab does not inhibit T-cell proliferation in a MLR (Vos et al., 201 1 , Gastroenterology, 140: 221 ).
  • EP2623515 A1 discloses humanized anti-TNFa antibodies and antigen-binding fragments (Fab) thereof. As becomes clear from the disclosed examples, the potency of the resulting humanized Fab fragments is comparable to that of infliximab in a L929 neutralization assay (see Table 2 and 5).
  • the sole anti-TNFa IgG antibody tested for cross-reactivity binds only weakly to Rhesus TNF-a (see [0069]; Fig. 3). Cross-reactivity with Cynomolgus TNFa was not tested. Moreover, there is weak binding to human TNFp (see Fig. 3).
  • EP2623515 A1 does not disclose anti-TNFa antibodies or functional fragments thereof, which have a potency to inhibit TNFa-induced apoptosis in L929 cells greater than that of infliximab and which are cross-reactive with Rhesus TNFa and Cynomolgus TNFa.
  • SDAB modified single domain antigen binding molecule
  • TNFa targets
  • linker e.g. TNFa
  • PEGylation a C-terminal Cysteine supporting the site specific PEGylation
  • WO 2012/007880 A2 fails to compare the potency of SDAB-01 to known TNFa antibodies like infliximab in a L929 cell-based neutralization assay, or to assess other SDAB-01 -specific parameters like the effectiveness to block TNFa -TNFRI/II interaction and the selectivity for binding TNFa over TNFp.
  • the treatment with SDAB-01 and infliximab are compared in a transgenic mouse model for polyarthritis that overexpresses human TNFa (see page 54, Example 8), the two seem to be similarly effective in preventing further development of arthritis (e.g. Fig. 17&18).
  • WO 2012/007880 A2 does not disclose anti- TNFa antibodies having a potency to inhibit TNFa-induced apoptosis in L929 cells greater than that of infliximab.
  • WO 2015/144852 A1 investigates the properties of an anti-TNF-a scFv designated “scFvl ".
  • This scFv showed a TNFa neutralization capacity in a PK-15 cell assay that was comparable to that of infliximab (see [0236]).
  • the scFv seems to have some cross-reactivity to TNF-a from rhesus macaque and cynomolgus monkey (see Ex. 8).
  • No affinity data are reported in WO 2015/144852 A1 .
  • the single chain antibody fragment DLX105 also known as ESBA 105
  • K D 157 pM; see Urech et al.
  • WO 2015/144852 A1 does not disclose anti-TNF-a antibodies having high affinity for human TNFa (K D ⁇ 125 pM).
  • WO 2015/065987 A1 describes anti-TNF-a antibodies, anti-IL-6 antibodies, and bispecific antibodies binding to both antigens. Certain anti-TNFa antibodies showed some cross- reactivity with TNFa from Cynomolgus (Fig. 17).
  • WO 2015/065987 A1 does not disclose anti-TNF- ⁇ antibodies having a potency to inhibit TNFa-induced apoptosis in L929 cells greater than that of infliximab.
  • a K D ⁇ 125 pM a high potency to inhibit TNFoinduced apoptosis in L929 cells, (iii) a high potency to inhibit LPS-induced cytokine secretion, (iv) substantial affinity to TNFa from Cynomolgus and Rhesus (e.g. a K D ⁇ 1 nM), and (v) a high melting temperature of the variable domain as determined in a thermal unfolding experiment (e.g. a T m > 70°C).
  • anti-TNFa antibodies and functional fragments thereof exhibit a combination of favorable properties, including high affinity for human TNFa (K D ⁇ 125 pM), a potency to inhibit TNFa-induced apoptosis in L929 cells greater than that of infliximab, a potency to inhibit LPS-induced cytokine secretion greater than that of adalimumab, and substantial affinity (K D ⁇ 1 nM) to TNFa from animals such as Cynomolgus monkey (Macaca fascicularis) and/or Rhesus macaques (Macaca mulatta).
  • the antibodies and functional fragments thereof were specific for TNFa in that they did not significantly bind to TNF3, and exhibit a significant stability, as determined in a thermal unfolding assay of the variable domain.
  • the invention provides antibody molecules and functional fragments thereof.
  • the present invention therefore relates to the subject matter defined in the following items (1 ) to (48):
  • CDR2 region having an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:2, and a CDR3 region having an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:3, and (ii) a V H domain comprising a CDR1 region having an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:4, a CDR2 region having an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:5, and a CDR3 region having an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:6.
  • antibody or functional fragment of any one of the preceding items wherein said antibody or functional fragment comprises a V L domain having an amino acid sequence selected from SEQ ID NO:14 and SEQ ID NO:54, preferably having the amino acid sequence as shown in SEQ ID NO:14.
  • (iii) has a greater potency than infliximab, as determined by an L929 assay; and/or (iv) is capable of binding to human TNFa Tr imer in a stoichiometry (antibody : TNFciTrimer) of at least 2.
  • the potency of the antibody or functional fragment to block the interaction between human TNFa and TNF receptor II (TNFRII), relative to that of infliximab (relative potency), as determined in an inhibition ELISA is at least 2, wherein said relative potency is the ratio of the IC 50 value in ng/mL of infliximab to the IC 50 value in ng/mL of the antibody in scFv format.
  • the antibody or functional fragment of any one of the preceding items which is capable of inhibiting cell proliferation of peripheral blood mononuclear cells in a mixed lymphocyte reaction.
  • the antibody or functional fragment of any one of the preceding items which is capable of inhibiting LPS-induced secretion of interleukin-1 ⁇ from CD14 + monocytes.
  • the antibody or functional fragment of any one of the preceding items which is capable of inhibiting LPS-induced secretion of TNFa from CD14 + monocytes.
  • the antibody of any one of the preceding items which is an immunoglobulin G
  • framework regions I to I I I of the variable light domain of said antibody or functional fragment consist of human VK1 consensus sequences with SEQ I D NOs:56 to 58, respectively, and framework region IV consists of a ⁇ germline-based sequence selected from SEQ I D NOs:59 to 62.
  • a vector or plasmid comprising the nucleic acid of item (34).
  • a cell comprising the nucleic acid of item (35) or the vector or plasmid of item (34).
  • a method of preparing the antibody or functional fragment of any one of items (1 ) to (33), comprising culturing the cell of item (36) in a medium under conditions that allow expression of the nucleic acid encoding the antibody or functional fragment, and recovering the antibody or functional fragment from the cells or from the medium.
  • a pharmaceutical composition comprising the antibody or functional fragment of any one of items (1 ) to (33), and optionally a pharmaceutically acceptable carrier and/or excipient. (39) The antibody or functional fragment as defined in any one of items (1 ) to (33) for use in a method of treating an inflammatory disorder or a TNForelated disorder.
  • ulcerative colitis The antibody or functional fragment for use according to item (41 ) or (42), wherein said inflammatory disorder of the gastrointestinal tract is ulcerative colitis.
  • said ulcerative colitis is selected from the group consisting of ulcerative proctitis, proctosigmoiditis, left-sided colitis, pan-ulcerative colitis, and pouchitis.
  • the antibody or functional fragment for use according to any one of items (39) to (47), wherein said method comprises orally administering the antibody or functional fragment to a subject.
  • Figure 1 Schematic representation of the humanization process.
  • FIG. 2 SE-HPLC chromatograms of purified humanized scFv preparations of an scFv.
  • the scFv monomer elutes at retention times between 8.5 and 9.5 minutes, while buffer components elute at >10 min. All peaks from the dead volume of the column up to the respective scFv monomer were integrated as aggregates/oligomers and used for the calculation of the relative peak area.
  • Figure 3 Thermal unfolding curves from DSF measurements of two scFv constructs. For each construct duplicate measurements are shown. The resulting Tm values have been determined by fitting the data to a Boltzmann equation to obtain the midpoint of transition.
  • Figure 4 Time-course of the monomer content of the two scFv constructs during storage.
  • the monomer content as determined by SE-HPLC has been plotted for the storage temperatures 4, -20 and ⁇ -65°C for the duration of 4 weeks.
  • Figure 5 Overlay of SE-HPLC chromatograms for two scFv molecules. For each scFv the sample (10 mg/ml) at dO and after storage for 4 weeks at 4°C is shown. In addition, the chromatogram of the sample after 5 cycles of freezing and thawing is shown. The inserted panel shows an approx. 15-fold zoom of the y-axis for each molecule to visualize also minuscule changes in oligomer content.
  • Figure 6 Time-course of the monomer content of the humanized scFvs during storage. The monomer content as determined by SE-HPLC has been plotted for the 10 mg/mL samples at a storage temperature of 37°C for the duration of 4 weeks.
  • Figure 7 Potency to neutralize human TNFa in the L929 assay of two scFvs. Dose- response curves for the scFvs and the reference antibody infliximab are shown for each experiment. The highest scFv and infliximab concentrations as well as negative controls were set to 100% and 0% of growth.
  • Figure 8 Potency of two scFvs to neutralize non-human primate and human TNFa in the L929 assay. Dose-response curves for neutralization of human, Cynomolgus monkey and Rhesus monkey TNFa are shown. The highest scFv concentration and negative controls were set to 100% and 0% of growth.
  • Figure 9 Potency of two scFvs to block the TNFa-TNFRI interaction. Dose-response curves are shown. The highest scFv concentration and negative controls were set to 0% and 100% of binding of TNFa to TNFRI.
  • Figure 10 Potency of two scFvs to block the TNFa-TNFRII interaction. Dose-response curves are shown. The highest scFv concentration and negative controls were set to 0% and 100% of binding of TNFa to TNFRII.
  • FIG. 1 Target specificity of an scFv.
  • the potential to inhibit the interaction of biotinylated TNFa with the scFv by TNFa and TNFp was analyzed by competition ELISA. Dose-dependent effects of TNFa and TNFp are shown.
  • Figure 12 depicts the formation of 16-22-H5-scFv:TNFa complexes determined by SE- HPLC (Example 4).
  • Figure 13 depicts the simultaneous binding of two TNFa molecules to 16-22-H5-scDb determined by SPR (Example 5).
  • Figure 14A depicts the formation of 16-22-H5-lgG:TNFa complexes (Example 5).
  • Figure 14B depicts the formation of 16-22-H5-scDb:TNFa complexes (Example 5).
  • Figure 15 depicts the inhibition of cell proliferation in a MLR following anti-TNFa treatment. * : p ⁇ 0.05; ** : p ⁇ 0.01 compared to IgG control (Example 6).
  • Figure 16 shows the ability of the different antibody formats of 16-22-H5 and adalimumab to inhibit the LPS-induced secretion of I L-1 ⁇ (Figure 16A) and TNFa (Figure 16B) in monocytes in a dose-dependent manner (Example 7).
  • the present invention pertains to an antibody or a functional fragment thereof capable of binding to human TNFa.
  • an antibody is used as a synonym for "immunoglobulin” (Ig), which is defined as a protein belonging to the class IgG, IgM, IgE, IgA, or IgD (or any subclass thereof), and includes all conventionally known antibodies and functional fragments thereof.
  • Ig immunoglobulin
  • a "functional fragment" of an antibody/immunoglobulin is defined as antigen-binding fragment or other derivative of a parental antibody that essentially maintains one or more of the properties of such parental antibody referred to in items (1 ) to (30) herein above.
  • an "antigen-binding fragment" of an antibody/immunoglobulin is defined as fragment (e.g., a variable region of an IgG) that retains the antigen-binding region.
  • An "antigen-binding region" of an antibody typically is found in one or more hypervariable region(s) of an antibody, i.e., the CDR-1 , -2, and/or -3 regions.
  • Antigen-binding fragments" of the invention include the domain of a F(ab') 2 fragment and a Fab fragment.
  • “Functional fragments" of the invention include, scFv, dsFv, diabodies, triabodies, tetrabodies and Fc fusion proteins.
  • the F(ab') 2 or Fab may be engineered to minimize or completely remove the intermolecular disulphide interactions that occur between the CH1 and CL domains.
  • the antibodies or functional fragments of the present invention may be part of bi- or multifunctional constructs.
  • Preferred functional fragments in the present invention are scFv and diabodies.
  • An scFv is a single chain Fv fragment in which the variable light (“V L ”) and variable heavy (“V H ”) domains are linked by a peptide bridge.
  • a diabody is a dimer consisting of two fragments, each having variable regions joined together via a linker or the like (hereinafter referred to as diabody-forming fragments), and typically contain two V L s and two V H s.
  • Diabody-forming fragments include those consisting of V
  • the linker joining variable regions is not specifically limited, but preferably enough short to avoid noncovalent bonds between variable regions in the same fragment. The length of such a linker can be determined as appropriate by those skilled in the art, but typically 2-14 amino acids, preferably 3-9 amino acids, especially 4-6 amino acids.
  • the V L and V H encoded on the same fragment are joined via a linker short enough to avoid noncovalent bonds between the V L and V H on the same chain and to avoid the formation of single-chain variable region fragments so that dimers with another fragment can be formed.
  • the dimers can be formed via either covalent or noncovalent bonds or both between diabody-forming fragments.
  • diabody-forming fragments can be joined via a linker or the like to form single- chain diabodies (sc(Fv) 2 ).
  • sc(Fv) 2 single- chain diabodies
  • polymerized antibodies such as trimers or tetramers can also be prepared by joining three or more diabody-forming fragments.
  • the antibody or functional fragment of the invention specifically binds to TNFa.
  • an antibody or functional fragment thereof “specifically recognizes”, or “specifically binds to” human TNFa, when the antibody or functional fragment is able to discriminate between human TNFa and one or more reference molecule(s).
  • the IC 50 value for binding to each of the reference molecules is at least 1 ,000 times greater than the IC 50 value for binding to TNFa, particularly as described in Example 2, section 2.1.4.
  • binding is referring to the ability of the antibody or functional fragment to discriminate between human TNFa and an unrelated biomolecule, as determined, for example, in accordance with a specificity assay methods known in the art. Such methods comprise, but are not limited to, Western blots and ELISA tests. For example, a standard ELISA assay can be carried out. Typically, determination of binding specificity is performed by using not a single reference biomolecule, but a set of about three to five unrelated biomolecules, such as milk powder, BSA, transferrin or the like. In one embodiment, specific binding refers to the ability of the antibody or fragment to discriminate between human TNFa and human TNF3.
  • the antibody of the invention or the functional fragment of the invention comprises a V L domain and a V H domain.
  • the V L domain comprises a CDR1 region (CDRL1 ), a CDR2 region (CDRL2), a CDR3 region (CDRL3) and Framework regions.
  • the V H domain comprises a CDR1 region (CDRH 1 ), a CDR2 region (CDRH2), a CDR3 region (CDRH3) and Framework regions.
  • CDR refers to one of the six hypervariable regions within the variable domains of an antibody that mainly contribute to antigen binding.
  • One of the most commonly used definitions for the six CDRs was provided by Kabat E. A. et al., (1991 ) Sequences of proteins of immunological interest.
  • CDR H1 or H1 is defined by the following residues (Kabat numbering): It starts with position 26 and ends prior to position 36.
  • the CDR1 region of the V L domain consists of an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:1 .
  • the CDR1 region of the V L domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22.
  • _ domain consists of the amino acid sequence as shown in SEQ ID NO:7.
  • the CDR2 region of the V L domain consists of an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:2.
  • the CDR2 region of the V L domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26.
  • the CDR2 region of the V L domain consists of the amino acid sequence as shown in SEQ ID NO:8.
  • the CDR3 region of the V L domain consists of an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:3.
  • the CDR3 region of the V L domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32.
  • the CDR3 region of the V L domain consists of the amino acid sequence as shown in SEQ ID NO:9.
  • the CDR1 region of the V H domain consists of an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:4.
  • the CDR1 region of the V H domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.
  • the CDR1 region of the V H domain consists of the amino acid sequence as shown in SEQ ID NO:10.
  • the CDR2 region of the V H domain consists of an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:5.
  • the CDR2 region of the V H domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 1 , SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41.
  • the CDR2 region of the V H domain consists of the amino acid sequence as shown in SEQ ID NO:1 1.
  • the CDR3 region of the V H domain consists of an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:6.
  • the CDR3 region of the V H domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48.
  • the CDR3 region of the V H domain consists of the amino acid sequence as shown in SEQ ID NO:12.
  • the antibody of the invention or the functional fragment of the invention comprises (i) a V L domain comprising a CDR1 region having an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:1 , a CDR2 region having an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:2, and a CDR3 region having an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:3, and (ii) a V H domain comprising a CDR1 region having an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:4, a CDR2 region having an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:5, and a CDR3 region having an amino acid sequence in accordance with the amino acid sequence as shown in SEQ ID NO:6
  • the antibody of the invention or the functional fragment of the invention comprises (i) a V L domain comprising a CDR1 region having the amino acid sequence as shown in SEQ ID NO:7, a CDR2 region having the amino acid sequence as shown in SEQ ID NO:8, and a CDR3 region having the amino acid sequence as shown in SEQ ID NO:9, and (ii) a V H domain comprising a CDR1 region having the amino acid sequence as shown in SEQ ID NO:10, a CDR2 region having the amino acid sequence as shown in SEQ ID NO:1 1 , and a CDR3 region having the amino acid sequence as shown in SEQ ID NO:12.
  • the antibody of the invention or the functional fragment of the invention comprises a V H domain having the amino acid sequence as shown in SEQ ID NO:13.
  • the antibody or functional fragment comprises a V L domain having the amino acid sequence as shown in SEQ ID NO:14 or SEQ ID NO:54.
  • the antibody of the invention or the functional fragment of the invention comprises (i) a V H domain having the amino acid sequence as shown in SEQ ID NO:13, and (ii) a V L domain having the amino acid sequence as shown in SEQ ID NO:14.
  • the functional fragment is a single chain antibody (scFv) comprising a V H domain having the amino acid sequence as shown in SEQ ID NO:13 and a V L domain having the amino acid sequence as shown in SEQ ID NO:14 or SEQ ID NO:54.
  • the V H domain and the V L domain are preferably linked by a peptide linker.
  • the peptide linker (hereinafter referred to as "linkerA”) typically has a length of about 10 to about 30 amino acids, more preferably of about 15 to about 25 amino acids.
  • the linkerA typically comprises Gly and Ser residues, but other amino acids are also possible.
  • the linker comprises multiple repeats of the sequence GGGGS (SEQ ID NO:50), e.g.
  • the linkerA consists of the amino acid sequence as shown in SEQ ID NO:49.
  • the scFv may have the following structure (with the N-terminus being left and the C-terminus being right):
  • _-LinkerA-V H or V H -LinkerA-V L .
  • the functional fragment is a single chain antibody (scFv) consisting of the amino acid sequence as shown in SEQ ID NO:15 or SEQ ID NO:55.
  • the functional fragment is a single chain antibody (scFv) consisting of the amino acid sequence as shown in SEQ ID NO:15.
  • the functional fragment is a diabody comprising a V H domain having the amino acid sequence as shown in SEQ ID NO:13 and a V
  • the V H domain and the V L domain are linked by a peptide linker.
  • the peptide linker (hereinafter referred to as "linkerB”) preferably has a length of about 2 to about 10 amino acids, more preferably of about 5 amino acids.
  • the linkerB typically comprises Gly and Ser residues, but other amino acids are also possible.
  • the linkerB consists of the amino acid sequence as shown in SEQ ID NO:50.
  • the diabody preferably is a monospecific diabody, i.e. it is directed to one epitope only.
  • the diabody is preferably a homodimer.
  • the diabody may be a dimer of two polypeptide chains that are non-covalently bound to each other. Each monomer may be a polypeptide chain having the structure: V L -LinkerB-V H ; or
  • V H -LinkerB-V L V H -LinkerB-V L .
  • diabody-forming fragments can be joined via a linkerA or the like to form single- chain diabodies (sc(Fv) 2 ).
  • linkerA single- chain diabodies
  • noncovalent bonds can be formed between diabody-forming fragments existing on the same chain to form dimers. Examples of the arrangements of single-chain diabodies include the following.
  • the diabody of the invention has the following structure: V L - linkerB - V H - linkerA - V L - linkerB - V H
  • the diabody consists of the amino acid sequence as shown in SEQ ID NO:51 .
  • polymerized antibodies such as trimers or tetramers can also be prepared by joining three or more diabody-forming fragments.
  • the antibody of the invention is an immunoglobulin, preferably an immunoglobulin G (IgG).
  • the subclass of the IgG of the invention is not limited and includes IgG-i , lgG 2 , lgG 3 , and lgG 4 .
  • the IgG of the invention is of subclass 1 , i.e. it is an Igd molecule.
  • each light chain of the IgG molecule of the invention has the amino acid sequence as shown in SEQ ID NO:52, and/or each heavy chain of the IgG molecule of the invention has the amino acid sequence as shown in SEQ ID NO:53.
  • a specific IgG of the invention consists of two light chains and two heavy chains, wherein each of the two light chains has the amino acid sequence as shown in SEQ ID NO:52, and each of the two heavy chains has the amino acid sequence as shown in SEQ ID NO:53.
  • the antibody or functional fragment of the invention has a high affinity to human TNFa.
  • K D refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.
  • the antibody or functional fragment of the invention binds to human TNFa with a dissociation equilibrium constant (K D ) of less than approximately 2x10 "10 M, preferably less than 1.5x10 "10 M, preferably less than 1.25x10 "10 M, more preferably less than 1 x10 "10 M, most preferably less than 7.5x10 "11 M or even less than 5x10 "11 M, as determined using surface plasmon resonance (SPR) technology in a BIACORE instrument.
  • SPR surface plasmon resonance
  • the antibody or functional fragment of the invention has substantial affinity to TNFa from animals such as Cynomolgus monkeys (Macaca fascicularis) and/or Rhesus macaques (Macaca mulatta). This is advantageous, as preclinical tests of anti-human TNFa antibodies such as toxicity studies are preferably performed with such animals.
  • the antibody or functional fragment of the invention is preferably cross-reactive with TNFa from animals such as Cynomolgus monkeys and/or Rhesus macaques. Affinity measurements are carried out as described in Example 2, section 2.1 .1 .
  • the antibody or functional fragment of the invention is cross-reactive with TNFa from Macaca fascicularis.
  • the antibody or functional fragment of the invention preferably has an affinity to Macaca fascicularis TNFa that is less than 20-fold, particularly less than 10-fold, even more particularly less than 5-fold different to that of human TNFa.
  • the antibody or functional fragment of the invention binds to TNFa from Macaca fascicularis with a dissociation equilibrium constant (K D ), wherein the ratio Riv asdcuiaris of (i) the K D for binding to TNFa from Macaca fascicularis to (ii) the K D for binding to human TNFa is less than 20.
  • RM.fascicuiaris is preferably less than 20, particularly less than 10, even more particularly less than 5.
  • the antibody or functional fragment of the invention is cross- reactive with TNFa from Macaca mulatta.
  • the antibody or functional fragment of the invention preferably has an affinity to Macaca mulatta TNFa that is less than 20-fold, more particularly less than 10-fold different to that of human TNFa.
  • the antibody or functional fragment of the invention binds to TNFa from Macaca mulatta with a dissociation equilibrium constant (K D ), wherein the ratio RM.muiatta of (i) the K D for binding to TNFa from Macaca mulatta to (ii) the K D for binding to human TN Fa is less than 20.
  • RM.muiatta is preferably less than 20, particularly less than 10.
  • the antibody or functional fragment of the invention is cross- reactive with TNFa from Macaca fascicularis and with TNFa from Macaca mulatta.
  • the antibody or functional fragment of the invention preferably has an affinity to Macaca fascicularis TNFa that is less than 20-fold, particularly less than 10-fold, even more particularly less than 5-fold different to that of human TN Fa, and it preferably has an affinity to Macaca mulatta TNFa that is less than 20-fold, more particularly less than 10-fold different to that of human TNFa.
  • the ratio Riv ascicuiaris of the antibody or functional fragment is preferably less than 20, particularly less than 10, even more particularly less than 5, and the ratio RM.muiatta of the antibody or functional fragment is preferably less than 20, particularly less than 10.
  • the antibody or functional fragment of the invention has a high potency to inhibit TNFa- induced apoptosis of L929 cells.
  • the antibody or functional fragment of the invention has a potency to inhibit TNFa-induced apoptosis of L929 cells greater than that of the known antibody infliximab.
  • Potency relative to infliximab can be determined in an L929 assay as described in Example 2, section 2.1 .2 of this application.
  • the relative potency of the antibody or functional fragment of the invention is greater than 1 .5, preferably greater than 2, more preferably greater than 3, more preferably greater than 5, more preferably greater than 7.5, or even greater than 10, wherein the relative potency is the ratio of (i) the IC 50 value of infliximab in an L929 assay over (ii) the IC 50 value of the antibody or functional fragment of the invention in the L929 assay, and wherein the IC 50 indicates the concentration in ng/mL of the respective molecule necessary to achieve 50% of maximal inhibition of TNFa-induced apoptosis of L929 cells.
  • the relative potency of the antibody or functional fragment of the invention is greater than 1 .5, preferably greater than 2, more preferably greater than 3, more preferably greater than 5, more preferably greater than 7.5, or even greater than 10, wherein the relative potency is the ratio of (i) the IC 90 value of infliximab in an L929 assay over (ii) the IC 90 value of the antibody or functional fragment of the invention in the L929 assay, and wherein the IC 90 value indicates the concentration in ng/mL of the respective molecule necessary to achieve 90% of maximal inhibition of TNFa-induced apoptosis of L929 cells .
  • the antibody or functional fragment of the invention is capable of inhibiting LPS- induced cytokine secretion from monocytes.
  • LPS-induced cytokine secretion from monocytes can be determined as described in Example 7.
  • the antibody or functional fragment of the invention is capable of inhibiting LPS-induced secretion of interleukin-1 ⁇ from CD14 + monocytes.
  • the IC 50 value for inhibiting LPS-induced secretion of interleukin-1 ⁇ is preferably less than 1 nM and/or less than 100 pg/mL.
  • the IC 50 value for inhibiting LPS-induced secretion of interleukin-1 ⁇ , on a molar basis and/or on a weight-per-volume basis, is preferably lower than that of adalimumab.
  • the antibody or functional fragment of the invention is capable of inhibiting LPS-induced secretion of TNFa from CD14 + monocytes.
  • the IC 50 value for inhibiting LPS-induced secretion of TNFa is preferably less than 1 nM and/or less than 150 pg/mL.
  • the IC 50 value for inhibiting LPS-induced secretion of TNFa, on a molar basis and/or on a weight-per-volume basis, is preferably lower than that of adalimumab.
  • the antibody or functional fragment of the invention is typically capable of inhibiting cell proliferation of peripheral blood mononuclear cells in a mixed lymphocyte reaction.
  • the inhibition of cell proliferation can be determined as described in Example 6.
  • the stimulation index of the antibody or functional fragment, e.g. of the scFv or diabody of the invention, determined according to Example 6, is preferably less than 5, more preferably less than 4.5.
  • the stimulation index of the antibody, e.g. of the IgG of the invention is less than 4 or even less than 3. Inhibition of interaction between TNFa and TNF receptor
  • the antibody or functional fragment of the invention is capable of inhibiting the interaction between human TNFa and TNF receptor I (TNFRI).
  • the inhibition of the interaction between human TNFa and TNFRI can be determined in an inhibition ELISA as described below in Example 2, section 2.1 .3.
  • the potency of the antibody or functional fragment of the invention to inhibit the interaction between human TNFa and TNFRI, relative to that of infliximab (relative potency), as determined in an inhibition ELISA is preferably at least 2, wherein said relative potency is the ratio of the IC 50 value in ng/mL of infliximab to the IC 50 value in ng/mL of the antibody or functional fragment thereof.
  • the antibody or functional fragment of the invention is capable of inhibiting the interaction between human TNFa and TNF receptor II (TNFRII).
  • TNFa and TNFRII TNF receptor II
  • the inhibition of the interaction between human TNFa and TNFRII can be determined in an inhibition ELISA as described below in Example 2, section 2.1 .3.
  • the potency of the antibody or functional fragment of the invention to inhibit the interaction between human TNFa and TNFRII, relative to that of infliximab (relative potency), as determined in an inhibition ELISA, is preferably at least 2, more preferably at least 3, wherein said relative potency is the ratio of the IC 50 value in ng/mL of infliximab to the IC 50 value in ng/mL of the antibody or functional fragment thereof.
  • the antibody or functional fragment of the invention is typically capable of binding to human TNFa T rimer in a stoichiometry (antibody : TNFa Tr imer) of at least 2.
  • the stoichiometry (antibody : TNFa Tr imer) is preferably greater than 2, or at least 2.5, or at least 3. In one embodiment, the stoichiometry (antibody : TNFa Tr imer) is about 3.
  • the stoichiometry (antibody : TNFa Tr imer) can be determined as described in Example 4 below.
  • the antibody or functional fragment of the invention is capable of forming a complex with human TNFa, wherein said complex comprises at least two molecules of TNFa and at least three molecules of antibody or functional fragment.
  • the functional fragment in accordance with this embodiment comprises at least two separate binding sites for TNFa such as, e.g. diabodies. Complex formation can be determined as described in Example 5 below.
  • the antibody is an IgG, and is capable of forming a complex of at least 600 kDa with TNFa.
  • the functional fragment is a diabody, and is capable of forming a complex of at least 300 kDa with TNFa.
  • the antibody or the functional fragment of the invention has a high target selectivity, i.e. it can discriminate between TNFa and TNF3.
  • the IC 50 value of TNF3 is at least 1 ,000 times greater than the IC 50 value of TNFa, as determined in a competition ELISA as described in Example 2, section 2.1 .4. More preferably, the IC 50 value of TNF3 is at least 5,000 times, most preferably at least 10,000 greater than the IC 50 value of TNFa, as determined in a competition ELISA as described in Example 2, section 2.1 .4.
  • the antibody or functional fragment of the invention preferably the scFv or diabody
  • the expression yield in E. coli, determined as described in Example 2 is at least 0.25 g/L. This particularly applies to functional fragments such as scFvs.
  • the refolding yield determined as described in Example 2, is at least 5 mg/L, more preferably at least 10 mg/L, more preferably at least 15 mg/L, and most preferably at least 20 mg/L. This particularly applies to functional fragments such as scFvs.
  • the antibody or functional fragment of the invention preferably the scFv or diabody, has a high stability. Stability can be assessed by different methodologies.
  • the "melting temperature" T m of the variable domain of the antibody or functional fragment of the invention determined by differential scanning fluorimetry (DSF) as described in Example 2, section 2.2.4, is preferably at least 65°C, more preferably at least 68°C, most preferably at least 70°C.
  • the "melting temperature of the variable domain” refers to the melting temperature of an scFv consisting of the sequence V L - LinkerA - V H , wherein the amino acid sequence of LinkerA consists of the amino acid sequence as shown in SEQ ID NO:49.
  • the melting temperature of the variable domain of an IgG is defined as the melting temperature of its corresponding scFv as defined above.
  • the loss in monomer content (at a concentration of 10 g/L; initial monomer content > 95%) after storage for four weeks at 4°C, determined by analytical size-exclusion chromatography as described in Example 2, section 2.2.5, is preferably less than 5%, more preferably less than 3%, more preferably less than 1 %, most preferably less than 0.5%.
  • the loss in monomer content (at a concentration of 10 g/L; initial monomer content > 95%) after storage for four weeks at -20°C, determined by analytical size-exclusion chromatography as described in Example 2, section 2.2.5, is preferably less than 5%, more preferably less than 3%, more preferably less than 1 %, most preferably less than 0.5%.
  • Functional fragments include, but are not limited to, F(ab') 2 fragment, a Fab fragment, scFv, diabodies, triabodies and tetrabodies.
  • the functional fragment is a single chain antibody (scFv) or a diabody. More preferably, the non-CDR sequences of the scFv or of the diabody are human sequences.
  • the chosen acceptor scaffold is composed of framework regions derived from human consensus sequences or human germline sequences.
  • framework regions I to III of the variable light domain consist of human VK1 consensus sequences according to SEQ ID NOs: 56 to 58 and a framework region IV of a ⁇ germline-based sequence selected from SEQ ID NOs:59 to 62.
  • residues that are not human consensus or human germline residues may cause immune reactions the number of such residues in each variable domain (VH or VL) should be as low as possible, preferably lower than 7, more preferably lower than 4, most preferably 0.
  • the antibody is a monoclonal antibody.
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. (Harlow and Lane, “Antibodies, A Laboratory Manual” CSH Press 1988, Cold Spring Harbor N.Y.).
  • the antibody is a human antibody or a humanized antibody, more preferably a monoclonal human antibody or a monoclonal humanized antibody.
  • chimeric antibody refers to an antibody having variable sequences derived from a non-human immunoglobulin, such as a rat or mouse antibody, and human immunoglobulins constant regions, typically chosen from a human immunoglobulin template.
  • Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719): 1202-7; Oi et al, 1986, BioTechniques 4:214- 221 ; Gillies et al., 1985, J. Immunol. Methods 125: 191 -202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entireties.
  • non-human antibody e.g., murine
  • immunoglobulins immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other target- binding subsequences of antibodies), which contain minimal sequences derived from such non-human immunoglobulin.
  • the resulting recombinant antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence, particularly a human immunoglobulin consensus sequence.
  • CDR-grafted antibodies are antibody molecules having one or more complementarity determining regions (CDRs) from an antibody originally generated in a non-human species that bind the desired antigen and framework (FR) regions from a human immunoglobulin molecule (EP239400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101 and 5,585,089).
  • framework residues in the human framework regions will additionally be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. See, e.g., Riechmann et al., 1988, Nature 332:323-7 and Queen et al, U.S. Patent Nos: 5,530,101 ; 5,585,089; 5,693,761 ; 5,693,762; and 6,180,370 (each of which is incorporated by reference in its entirety).
  • Antibodies can be rendered more human using a variety of additional techniques known in the art including, for example, veneering or resurfacing (EP592106; EP519596; Padlan, 1991 , Mol. Immunol, 28:489-498; Studnicka et al, 1994, Prot. Eng. 7:805-814; Roguska et al, 1994, Proc. Natl. Acad. Sci. 91 :969-973, and chain shuffling (U.S. Patent No. 5,565,332), all of which are hereby incorporated by reference in their entireties.
  • a CDR-grafted or humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin template chosen.
  • Fc immunoglobulin constant region
  • humanized antibodies are prepared as described in Queen et al, U.S. Patent Nos: 5,530,101 ; 5,585,089; 5,693,761 ; 5,693,762; and 6,180,370 (each of which is incorporated by reference in its entirety).
  • the anti-TNFa antibodies are human antibodies.
  • Completely "human” anti-TNFa antibodies can be desirable for therapeutic treatment of human patients.
  • "human antibodies” include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,1 1 1 ; and PCT publications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741 , each of which is incorporated herein by reference in its entirety. Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • Completely human antibodies that recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g., a mouse antibody
  • is used to guide the selection of a completely human antibody recognizing the same epitope Jespers et al, 1988, Biotechnology 12:899-903.
  • the anti-TNFa antibodies are primatized antibodies.
  • the term "primatized antibody” refers to an antibody comprising monkey variable regions and human constant regions. Methods for producing primatized antibodies are known in the art. See e.g., U.S. Patent Nos. 5,658,570; 5,681 ,722; and 5,693,780, which are incorporated herein by reference in their entireties.
  • the anti-TNFa antibodies are derivatized antibodies.
  • the derivatized antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein (see below for a discussion of antibody conjugates), etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • an anti-TNFa antibody has one or more amino acids inserted into one or more of its hypervariable region, for example as described in US 2007/0280931 .
  • the anti-TNFa antibodies are antibody conjugates that are modified, e.g., by the covalent attachment of any type of molecule to the antibody, such that covalent attachment does not interfere with binding to TNFa.
  • Techniques for conjugating effector moieties to antibodies are well known in the art (See, e.g., Hellstrom et al., Controlled Drag Delivery, 2nd Ed., at pp. 623-53 (Robinson et al., eds., 1987)); Thorpe et al., 1982, Immunol. Rev. 62: 1 19-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics 83:67-123).
  • the antibody or fragment thereof is fused via a covalent bond (e.g., a peptide bond), at optionally the N-terminus or the C-terminus, to an amino acid sequence of another protein (or portion thereof; preferably at least a 10, 20 or 50 amino acid portion of the protein).
  • a covalent bond e.g., a peptide bond
  • the antibody, or fragment thereof is linked to the other protein at the N- terminus of the constant domain of the antibody.
  • Recombinant DNA procedures can be used to create such fusions, for example as described in WO 86/01533 and EP0392745.
  • the effector molecule can increase half-life in vivo.
  • anti-TNFa antibodies can be attached to poly(ethyleneglycol) (PEG) moieties.
  • PEG poly(ethyleneglycol)
  • the PEG moieties can be attached through any available amino acid side-chain or terminal amino acid functional group located in the antibody fragment, for example any free amino, imino, thiol, hydroxyl or carboxyl group.
  • amino acids can occur naturally in the antibody fragment or can be engineered into the fragment using recombinant DNA methods. See for example U.S. Patent No. 5,219,996. Multiple sites can be used to attach two or more PEG molecules.
  • PEG moieties are covalently linked through a thiol group of at least one cysteine residue located in the antibody fragment.
  • a thiol group is used as the point of attachment
  • appropriately activated effector moieties for example thiol selective derivatives such as maleimides and cysteine derivatives, can be used.
  • an anti-TNFa antibody conjugate is a modified Fab' fragment which is PEGylated, i.e., has PEG (poly(ethyleneglycol)) covalently attached thereto, e.g., according to the method disclosed in EP0948544.
  • PEG poly(ethyleneglycol)
  • Treatment of a disease encompasses the treatment of patients already diagnosed as having any form of the disease at any clinical stage or manifestation; the delay of the onset or evolution or aggravation or deterioration of the symptoms or signs of the disease; and/or preventing and/or reducing the severity of the disease.
  • a "subject" or “patient” to whom an anti-TNFa antibody or functional fragment thereof is administered can be a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey or human).
  • a non-primate e.g., cow, pig, horse, cat, dog, rat, etc.
  • a primate e.g., monkey or human.
  • the human is a pediatric patient. In other aspects, the human is an adult patient.
  • compositions comprising an anti-TNFa antibody and, optionally one or more additional therapeutic agents, such as the second therapeutic agents described below, are described herein.
  • the compositions typically are supplied as part of a sterile, pharmaceutical composition that includes a pharmaceutically acceptable carrier.
  • This composition can be in any suitable form (depending upon the desired method of administering it to a patient).
  • the anti-TNFa antibodies and functional fragments can be administered to a patient by a variety of routes such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intrathecally, topically or locally.
  • routes such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intrathecally, topically or locally.
  • the most suitable route for administration in any given case will depend on the particular antibody, the subject, and the nature and severity of the disease and the physical condition of the subject.
  • an anti-TNFa antibody or functional fragment thereof will be administered intravenously.
  • the antibody or functional fragment of the invention is administered orally. If the administration is via the oral route the functional fragment is preferably a single chain antibody (scFv), diabody or IgG.
  • an anti-TNFa antibody or functional fragment is present in a pharmaceutical composition at a concentration sufficient to permit intravenous administration at 0.5 mg/kg body weight to 20 mg/kg body weight.
  • the concentration of antibody or fragment suitable for use in the compositions and methods described herein includes, but is not limited to, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 1 1 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, or a concentration ranging between any of the foregoing values, e.g., 1 mg/kg to 10 mg/kg, 5 mg/kg to 15 mg/kg, or 10 mg/kg to 18 mg/kg.
  • the effective dose of an anti-TNFa antibody or functional fragment can range from about 0.001 to about 750 mg/kg per single (e.g., bolus) administration, multiple administrations or continuous administration, or to achieve a serum concentration of 0.01 -5000 ⁇ g/ml serum concentration per single (e.g., bolus) administration, multiple administrations or continuous administration, or any effective range or value therein depending on the condition being treated, the route of administration and the age, weight and condition of the subject.
  • each dose can range from about 0.5 mg to about 50 mg per kilogram of body weight or from about 3 mg to about 30 mg per kilogram body weight.
  • the antibody can be formulated as an aqueous solution.
  • compositions can be conveniently presented in unit dose forms containing a predetermined amount of an anti-TNFa antibody or functional fragment per dose.
  • a unit can contain 0.5 mg to 5 g, for example, but without limitation, 1 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 750 mg, 1000 mg, or any range between any two of the foregoing values, for example 10 mg to 1000 mg, 20 mg to 50 mg, or 30 mg to 300 mg.
  • Pharmaceutically acceptable carriers can take a wide variety of forms depending, e.g., on the condition to be treated or route of administration.
  • an anti-TNFa antibody or functional fragment thereof is well within the capabilities of those skilled in the art, and can be determined using a standard dose escalation study.
  • Therapeutic formulations of the anti-TNFa antibodies and functional fragments suitable in the methods described herein can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the antibody having the desired degree of purity with optional pharmaceutically-acceptable carriers, excipients or stabilizers typically employed in the art (all of which are referred to herein as "carriers"), i.e., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives. See, Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives must be nontoxic to the recipients at the dosages and concentrations employed.
  • Buffering agents help to maintain the pH in the range which approximates physiological conditions. They can present at concentration ranging from about 2 mM to about 50 mM.
  • Suitable buffering agents include both organic and inorganic acids and salts thereof such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid- monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid- disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, mono
  • Preservatives can be added to retard microbial growth, and can be added in amounts ranging from 0.2%- 1 % (w/v).
  • Suitable preservatives include phenol, benzyl alcohol, meta- cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalconium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3- pentanol.
  • Isotonicifiers sometimes known as “stabilizers” can be added to ensure isotonicity of liquid compositions and include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
  • Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall.
  • Typical stabilizers can be polyhydric sugar alcohols (enumerated above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, omonothioglycerol and sodium thio sulfate; low mo
  • Stabilizers can be present in the range from 0.1 to 10,000 weights per part of weight active protein.
  • Non-ionic surfactants or detergents also known as "wetting agents" can be added to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stressed without causing denaturation of the protein.
  • Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.).
  • Non-ionic surfactants can be present in a range of about 0.05 mg/ml to about 1 .0 mg/ml, or in a range of about 0.07 mg/ml to about 0.2 mg/ml.
  • Additional miscellaneous excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), protease inhibitors and co-solvents.
  • bulking agents e.g., starch
  • chelating agents e.g., EDTA
  • antioxidants e.g., ascorbic acid, methionine, vitamin E
  • protease inhibitors e.g., ascorbic acid, methionine, vitamin E
  • the formulation herein can also contain a second therapeutic agent in addition to an anti- TNFa antibody or functional fragment thereof.
  • a second therapeutic agent in addition to an anti- TNFa antibody or functional fragment thereof. Examples of suitable second therapeutic agents are provided below.
  • the dosing schedule can vary from once a month to daily depending on a number of clinical factors, including the type of disease, severity of disease, and the patient's sensitivity to the anti-TNFa antibody or functional fragment.
  • an anti-TNFa antibody or functional fragment thereof is administered daily, twice weekly, three times a week, every other day, every 5 days, every 10 days, every two weeks, every three weeks, every four weeks or once a month, or in any range between any two of the foregoing values, for example from every four days to every month, from every 10 days to every two weeks, or from two to three times a week, etc.
  • the dosage of an anti-TNFa antibody or functional fragment to be administered will vary according to the particular antibody, the subject, and the nature and severity of the disease, the physical condition of the subject, the therapeutic regimen (e.g., whether a second therapeutic agent is used), and the selected route of administration; the appropriate dosage can be readily determined by a person skilled in the art. It will be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of an anti-TNFa antibody or functional fragment thereof will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the age and condition of the particular subject being treated, and that a physician will ultimately determine appropriate dosages to be used. This dosage can be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be altered or reduced, in accordance with normal clinical practice.
  • the invention relates to a method of treating or preventing a human TNFa-related disease in a subject, comprising administering to the subject the antibody or functional fragment as defined herein.
  • TNFa-related disorder or “TNFa-related disease” refers to any disorder, the onset, progression or the persistence of the symptoms or disease states of which requires the participation of TNFa.
  • TNFa-related disorders include, but are not limited to, chronic and/or autoimmune states of inflammation in general, immune mediated inflammatory disorders in general, inflammatory CNS disease, inflammatory diseases affecting the eye, joint, skin, mucous membranes, central nervous system, gastrointestinal tract, urinary tract or lung, states of uveitis in general, retinitis, HLA-B27+ uveitis, Behget's disease, dry eye syndrome, glaucoma, Sjogren syndrome, diabetes mellitus (incl.
  • diabetic neuropathy insulin resistance
  • states of arthritis in general rheumatoid arthritis, osteoarthritis, reactive arthritis and Reiter's syndrome, juvenile arthritis, ankylosing spondylitis, multiple sclerosis, Guillain-Barre syndrome, myasthenia gravis, amyotrophic lateral sclerosis, sarcoidosis, glomerulonephritis, chronic kidney disease, cystitis, psoriasis (incl.
  • extraintestinal manifestastations ulcerative colitis
  • asthma bronchiale hypersensitivity pneumonitis
  • general allergies allergic rhinitis, allergic sinusitis, chronic obstructive pulmonary disease (COPD)
  • COPD chronic obstructive pulmonary disease
  • lung fibrosis Wegener's granulomatosis
  • Kawasaki syndrome Giant cell arteritis
  • Churg-Strauss vasculitis polyarteritis nodosa, burns, graft versus host disease, host versus graft reactions, rejection episodes following organ or bone marrow transplantation, systemic and local states of vasculitis in general, systemic and cutaneous lupus erythematodes, polymyositis and dermatomyositis, sclerodermia, pre-eclampsia, acute and chronic pancreatitis, viral hepatitis, alcoholic hepatitis, postsurgical inflammation such as after eye surgery (e.g.
  • cataract eye lens replacement
  • glaucoma surgery joint surgery
  • joint-related structures e.g. ligaments
  • oral and/or dental surgery minimally invasive cardiovascular procedures (e.g. PTCA, atherectomy, stent placement), laparoscopic and/or endoscopic intra-abdominal and gynecological procedures, endoscopic urological procedures (e.g.
  • prostate surgery in general, ureteroscopy, cystoscopy, interstitial cystitis), or perioperative inflammation (prevention) in general, bullous dermatitis, neutrophilic dermatitis, toxic epidermal necrolysis, pustular dermatitis, cerebral malaria, hemolytic uremic syndrome, allograft rejection, otitis media, snakebite, erythema nodosum, myelodysplastic syndromes, primary sclerosing cholangitis, seronegative spondylartheropathy, autoimmune hematolytic anemia, orofacial granulamatosis, pyostomatitis vegetans, aphthous stomatitis, geographic tongue, migratory stoimatitis, Alzheimer disease, Parkinson's disease, Huntington's disease, Bell's palsy, Creutzfeld- Jakob disease and neuro-degenerative conditions in general.
  • Cancer-related osteolysis, cancer-related inflammation, cancer-related pain, cancer-related cachexia, bone metastases, acute and chronic forms of pain irrespective whether these are caused by central or peripheral effects of TNFa and whether they are classified as inflammatory, nociceptive or neuropathic forms of pain, sciatica, low back pain, carpal tunnel syndrome, complex regional pain syndrome (CRPS), gout, postherpetic neuralgia, fibromyalgia, local pain states, chronic pain syndroms due to metastatic tumor, dismenorrhea.
  • Particular disorders to be treated include states of arthritis in general, rheumatoid arthritis, osteoarthritis, reactive arthritis, juvenile arthritis; psoriasis incl.
  • psoriatic arthritis inflammatory bowel disease, including Crohn's disease, ulcerative colitis incl. proctitis, sigmoiditis, left-sided colitis, extensive colitis and pancolitis, undetermined colitis, microscopic colitis incl. collagenous and lymphocytic colitis, colitis in connective tissue disease, diversion colitis, colitis in diverticular disease, eosinophilic colitis and pouchitis.
  • the antibody or functional fragment of the invention is used to treat an inflammatory bowel disease, in particular Crohn's disease, ulcerative colitis or microscopic colitis.
  • the Crohn's disease may be ileal, colonic, ileocolonic or isolated upper Crohn's disease (gastric, duodenal and/or jejunal) including non-stricturing/non-penetrating, stricturing, penetrating and perianal disease behavior, allowing any combination of localization and disease behavior of any of the above mentioned.
  • the ulcerative colitis may be ulcerative proctitis, proctosigmoiditis, left-sided colitis, pan-ulcerative colitis and pouchitis. Combination Therapy and other aspects
  • the patient being treated with an anti-TNFa antibody or functional fragment thereof is also treated with another conventional medicament.
  • a patient suffering from inflammatory bowel disease, especially if having moderate to severe disease is typically also being treated with mesalazine or derivatives or prodrugs thereof, corticosteroids, e.g. budesonide or prednisolone (oral or i.v.), immunosuppressants, e.g. azathioprine/6-mercaptopurine (6-MP) or methotrexate, cyclosporine or tacrolimus.
  • corticosteroids e.g. budesonide or prednisolone (oral or i.v.
  • immunosuppressants e.g. azathioprine/6-mercaptopurine (6-MP) or methotrexate, cyclosporine or tacrolimus.
  • medicaments which can be co-administered to the patient include biologies such as infliximab, adalimumab, etanercept, certolizumab pegol or others. Further medicaments which can be co-administered to the patient include immunosupressants (e.g. azathioprine/6-MP or methotrexate or oral cyclosporine) in order to maintain stable and longer remission.
  • immunosupressants e.g. azathioprine/6-MP or methotrexate or oral cyclosporine
  • an anti-TNFa antibody or functional fragment as defined hereinabove for reducing inflammation.
  • an anti-TNFa antibody or functional fragment as defined hereinabove for use in reducing inflammation in a patient suffering from an inflammatory condition.
  • a further aspect of this invention is a method of treating an inflammatory condition, comprising administering to a patient in need thereof an effective amount of an anti-TNFa antibody or functional fragment as defined hereinabove.
  • the inflammatory condition is preferably one of the conditions described above.
  • a further aspect of this invention is a method of preventing an inflammatory condition, comprising administering to a patient in need thereof an effective amount of an anti-TNFa antibody or functional fragment as defined hereinabove.
  • the inflammatory condition is preferably one of the conditions described above.
  • Example 1 Generation of rabbit antibodies directed against human TNFa
  • Rabbits have been immunized with purified recombinant human TNFa (Peprotech, Cat. No. 300-01 A). During the course of the immunization, the strength of the humoral immune response against the antigen was qualitatively assessed by determining the maximal dilution (titer) for the serum of each rabbit that still produced detectable binding of the polyclonal serum antibodies to the antigen. Serum antibody titers against immobilized recombinant human TNFa were assessed using an enzyme-linked immunosorbent assay (ELISA, see 2.2.1 ).
  • ELISA enzyme-linked immunosorbent assay
  • mice showed very high titers with a 10 x 10 6 -fold dilution of the serum still resulting in a positive signal (at least 3-fold higher than the signal obtained with serum from a naive unrelated animal which was used as background control) in the ELISA.
  • the ability of different rabbit sera to inhibit the biological activity of TNFa was assessed using a mouse L929 cell-based assay (see 2.2.3). All three sera inhibited TN Fa-induced apoptosis of mouse L929 fibroblasts. Rabbit #3 showed the strongest neutralizing activity with 50% inhibition (IC 50 ) reached at a serum dilution of 1 :155'000.
  • rabbit #2 and rabbit #1 showed approximately 3 and 21 -fold lower activity, reaching 50% inhibition at a serum dilution of 1 :55'500 and 1 :7'210, respectively.
  • Lymphocytes isolated from spleens of all three animals were chosen for the subsequent hit identification procedures. The animals were prioritized based on the potency to inhibit the biological activity of TNFa in the L929 assay. Therefore, the highest number of hits that were cultivated originated from rabbit #3, and the lowest number of hits was derived from rabbit #1 .
  • a total of 33 x 10 6 lymphocytes (corresponding to 1 .5% of total lymphocytes isolated) derived from all three rabbits were characterized in two independent sorting campaigns. Out of the 33 x 10 6 cells analyzed in total 3452 B-cells expressing TNFa-specific antibodies (IgG) were isolated. The numbers of lymphocytes cloned were different for the three rabbits, as more cells were isolated from those rabbits whose sera showed strong inhibition of TNFa in the L929 assay. Of the isolated B-cells, 792 clones were derived from rabbit #1 , 1 144 clones from rabbit #2 and 1408 clones from rabbit #3. For 108 clones the respective rabbit origin is not known, because they are derived from a mixture of residual lymphocytes from all 3 rabbits to allow optimal recovery of small amount of lymphocytes from the vials.
  • results obtained during the screening phase are based on assays performed with non- purified antibodies from culture supernatants of antibody secreting cells (ASC), as the scale of the high-throughput culture does not allow for purification of the individual rabbit antibodies.
  • ASC antibody secreting cells
  • Such supernatants were used to rank large numbers of antibodies relative to each other, however not to provide absolute values (e.g. for inhibition of biological activity of TNFa), except for binding affinity.
  • ASC supernatants were screened in a high-throughput ELISA for binding to recombinant human TNFa.
  • TNFa-binding supernatants were further characterized for binding to Cynomolgus monkey TNFa by ELISA, binding kinetics and for their potential to neutralize the biological activity of human TNFa in the L929 assay. With the exception of binding kinetics, the reporting values of the high-throughput screenings should be interpreted as "yes” or "no" answers, which are based on single-point measurements (no dose-response). Affinity to Cynomolgus monkey and mouse TNFa was analyzed for all the 102 clones that were selected for amplification and sequencing of the antibody heavy and light chain variable domains.
  • the aim of the primary screening is to identify ASC clones that produce antibodies specific for human TNFa.
  • cell culture supernatants of 3452 ASC clones were analysed for the presence of antibodies to human TNFa by ELISA (see 2.2.1 ).
  • the ELISA method used assesses the "quantity" of antibodies of the IgG subtype bound to recombinant human TNFa, gives however no information about the affinity or the concentration of the antibodies.
  • supernatants from 894 ASC clones produced a signal that was clearly above background.
  • the hit rate in screening was similar for rabbit #1 and rabbit #2 with 153 hits out of 792 (19.3%) identified from rabbit #1 and 225 hits out of 1 144 identified from rabbit #2 (19.7%).
  • Rabbit #3 showed a significantly higher hit rate of 34.4% resulting in the identification of 484 hits out of 1408. All 894 hits identified in this primary screening, were subjected to the measurement of binding kinetics by SPR (secondary screening).
  • the aim of the secondary screening is to obtain quantitative information on the quality of target binding for each hit from the primary screening by surface plasmon resonance (SPR, see 2.2.2).
  • SPR surface plasmon resonance
  • this method assesses the kinetics of target binding as a function of time. This allows determination of the rate constants for association (k a ) and dissociation (k d ) of the antibody from its target.
  • the ratio k d /k a provides the equilibrium dissociation constant (K D ), which reflects the affinity of an antibody to its target.
  • binding affinities to human TNFa could be determined for 839 monoclonal rabbit antibodies.
  • the 839 anti-TNFa antibodies that could be measured showed dissociation constants (K D ) ranging from below 1 .36 x 10 "13 M to 1 .14 x 10 "8 M. 69% of all antibodies analyzed had a K D below 0.5 nM.
  • L929 assay For the assessment of potency, a cell-based assay (L929 assay) has been developed (see 2.2.3). 506 out of the 894 selected antibodies (56.6%), inhibited TNFa-induced apoptosis in the L929 assay by more than 50%. In line with results obtained during titer analysis, the highest percentage of neutralizing hits was derived from rabbit #3 with a hit rate of 62.8%, followed by rabbit #2 with a hit rate of 56.4% and rabbit #1 with the lowest hit rate of 39.9%. Affinities of these neutralizing antibodies ranged between 1 .36 x 10 "13 to 1.19 x 10 "9 M.
  • the genetic information encoding the rabbit antibody variable domain needs to be retrieved. This was done by reverse transcription (RT) of the respective messenger RNA into the complementary DNA (cDNA), followed by amplification of the double stranded DNA by the polymerase chain reaction (PCR).
  • RT reverse transcription
  • PCR polymerase chain reaction
  • the selection of ASC clones subjected to RT-PCR was primarily based on affinity and neutralizing activity. As additional criterion cross-reactivity to Cynomolgus monkey TNFa was considered. In total 102 ASC clones were selected for gene cloning by RT-PCR.
  • Table 2 Pharmacodynamic properties of monoclonal antibodies in ASC supernatants. .
  • ASC SN Affinity to hu ma n TNFa Affinity to Cynomolgus TNFa
  • TNFa Binding ELISA human and Cynomolgus monkey TNFa
  • Recombinant human TNFa (Peprotech, Cat. No. 300-01 ) was coated on a 96 well microtiter ELISA plate. Binding of rabbit antibodies in the ASC culture supernatants to the immobilized TNFa was detected by a secondary HRP-labelled anti-rabbit IgG (Jacksonlmmuno Research, Cat. No. 1 1 1 -035-046). TMB substrate (3,3',5,5'- tetramethylbenzidine, KPL, Cat. No. 53-00-00) was added and the colour reaction was stopped by the addition of H 2 S0 4 . Plates were read using a microtiter plate reader (Infinity reader M200 Pro, Tecan) at a wavelength of 450 nm.
  • Assay performance during the screening campaigns was monitored by a commercially available positive control anti-TNFa rabbit polyclonal antibody (AbD Serotec, Cat. No. 9295- 0174).
  • the positive control antibody was tested at 100 and 250 ng/ml in duplicate on each screening plate. Robustness and precision of the response of the positive control was monitored for each plate.
  • the signal-to-background ratio was between 30 to 40 for the positive control at 250 ng/ml and coefficient of variation (CV) of the positive control were below 10%.
  • a signal with an optical density of ⁇ 100% relative to the 250 ng/ml positive control was considered as a primary screening hit.
  • Binding affinities of antibodies towards human TNFa were measured by surface plasmon resonance (SPR) using a MASS-1 SPR instrument (Sierra Sensors). Performance of the instrument was qualified by means of standard reference solutions as well as by analysis of a reference antibody-antigen interaction such as infliximab-TNFa interaction.
  • an antibody specific for the Fc region of rabbit IgGs (Bethyl Laboratories, Cat. No. A120-1 1 1A) was immobilized on a sensor chip (SPR-2 Affinity Sensor, High Capacity Amine, Sierra Sensors) using a standard amine-coupling procedure.
  • Rabbit monoclonal antibodies in ASC supernatants were captured by the immobilized anti- rabbit IgG antibody.
  • human TNFa (Peprotech, Cat. No. 300-01 ) was injected into the flow cells for 3 min at a concentration of 90 nM, and dissociation of the protein from the IgG captured on the sensor chip was allowed to proceed for 5 min.
  • L929 fibroblasts ATCC/LGC Standards, Cat. No. CCL-1 .
  • L929 cells were sensitized to TNFa-induced apoptosis by addition of 1 g/ml actinomycin D.
  • Cells were cultured in 96-well flat-bottom microtiter plates in the presence of 50% AS C culture supernatant and 100 pM (5.2 ng/ml) human TNFa (Peprotech, Cat. No. 300-01 ) for 24 h.
  • the concentration of infliximab required to neutralize TNFa-induced apoptosis by 50% (IC 50 ) was calculated at a concentration of 36.2 ng/ml. Therefore, the estimated lower limit of detection for this assay is between 30 to 40 ng/ml. This value is only a rough estimate for the detection limit, since the potential to block TNFa is not only dependent on the concentration of the monoclonal antibody but also on affinity of the antibody to the target. However, the sensitivity of the assay is sufficient for screening of ASC supernatants since IgG concentrations in most ASC supernatants are above a concentration of 40 ng/ml.
  • 73 unique sets of parental rabbit light and heavy chain variable domains were retrieved during hit screening and analyzed by sequence alignment. Based on the screening assay results and the sequence homology of the individual rabbit IgG clones, 30 candidates were selected for hit confirmation. 29 monoclonal antibodies were manufactured and the best performing clones in terms of affinity and potency were selected for the humanization and lead candidate generation. The criteria for the selection of clones were i) neutralization of human TNFa in L929 assay, ii) high affinity to human TNFa, iii) cross-reactivity to Cynomolgus and Rhesus monkey TNFa, and iv) sequence diversity.
  • One clone (1 6-22-H05) has been selected for humanization - one of the best ranking IgGs in terms of potency to neutralize human TNFa in the L929 assay. With respect to binding strength, the best possible affinity is desired since a certain loss of affinity needs to be anticipated as a result of humanization and reformatting into the scFv format.
  • CDRs complementarity determining regions
  • the humanization of the selected clone comprised the transfer of the rabbit CDRs onto a scFv acceptor framework of the VK1 /VH3 type as described in WO 2014/206561 .
  • the amino acid sequence of the six CDR regions was identified on the donor sequence (rabbit mAb) and grafted into the acceptor scaffold sequence, resulting in the constructs termed "CDR graft".
  • a second graft was designed, which included additional amino acids modifications from the rabbit donor on the positions L15, L22, L48, L57, L74, L87, L88, L90, L92, L95, L97, L99 and H24, H25, H56, H82, H84, H89, H108 (AHo numbering), which have been described to potentially influence CDR positioning and thus antigen binding (Borras et al. JBC. 2010; 285:9054-9066).
  • STR structural
  • the heterologous expression of the proteins was performed in E.coli as insoluble inclusion bodies.
  • the expression culture was inoculated with an exponentially growing starting culture. The cultivation was performed in shake flasks in an orbital shaker using commercially available rich media. The cells were grown to a defined OD 6 oo of 2 and induced by overnight expression with 1 mM Isopropyl ⁇ -D-l -thiogalactopyranoside (IPTG). At the end of fermentation the cells were harvested by centrifugation and homogenized by sonication. At this point the expression level of the different constructs was determined by SDS-PAGE analysis of the cell lysate.
  • the inclusion bodies were isolated from the homogenized cell pellet by a centrifugation protocol that included several washing steps to remove cell debris and other host cell impurities.
  • the purified inclusion bodies were solubilized in a denaturing buffer (100 mM Tris/HCI pH 8.0, 6 M Gdn-HCI, 2 mM EDTA) and the scFvs were refolded by a scalable refolding protocol that generated milligram amounts of natively folded, monomeric scFv.
  • a standardized protocol was employed to purify the scFvs, which included the following steps.
  • the product after refolding was captured by an affinity chromatography employing Capto L agarose (GE Healthcare) to yield the purified scFvs.
  • the scFv was investigated as to certain criteria, as explained in the following.
  • the producibility criterion shall ensure that the selected scFv entity can be expressed, refolded and purified in sufficient amounts to support later development of the lead molecule.
  • the defined criteria were the expression yield of scFv per liter of fermentation broth, as assessed by SDS-PAGE, and the purification yield achieved in the generic lab- scale process, as assessed by measurement of the amount of purified protein by UV spectrometry, calculated back to 1 liter of refolding solution.
  • the criteria for stability were intended to assess the aggregation propensity during the manufacturing process of the molecules and their structural integrity during storage and further handling.
  • the monomer content determined by SE-HPLC allows assessing the colloidal stability of molecules during the purification process (2.2.3).
  • the monomer content was tested over a duration of 4 weeks at 1 and 10 mg/mL and storage at 4, -20 and ⁇ -65°C.
  • the colloidal stability of the proteins was tested after 5 freezing and thawing cycles.
  • the midpoint of thermal unfolding was determined by differential scanning fluorimetry (DSF) (2.2.4) to provide a read-out for the conformational stability of the lead candidates.
  • DSF differential scanning fluorimetry
  • the lead candidate scFv molecules were expressed by shake flask fermentation in batch mode and purified by a generic lab-scale process to yield the protein samples for further characterization. During this process some key performance parameters were monitored to compare the candidate molecules and to identify potentially difficult to develop constructs.
  • the expression titer was determined on the level of the crude E.coli lysate after the harvest of the cells by centrifugation. During the harvest a small loss of cells is anticipated, however, this factor was chosen to be neglected for the calculation of the expression yield in favor of a more conservative estimation of the productivity.
  • coomassie stained reducing SDS-PAGE (2.2.1 ) was chosen due to the high specificity of the method that allows discriminating the product from the host cell proteins in the sample.
  • a second criterion to assess the producibility is the purification yield of scFv calculated per liter of refolding solution. This parameter addresses the potential bottleneck in the anticipated manufacturing process that includes a protein refolding step. Since the efficiency of the refolding procedure has proven to be limiting in comparable manufacturing processes it has been decided to compare the performance of the different constructs with respect to the producibility normalized to a defined refolding volume. For the calculation of the yield the final protein sample from each batch was quantified by UV absorbance (2.2.2) and divided by the actual refolding volume of the respective purification (Table 6). Table 6: Summary of producibility data for two humanized scFvs.
  • the expression titer was determined by quantitative SDS-PAGE on lysates of end-of-production cells.
  • the batch yield was determined by UV absorbance measurement of the final purification pool.
  • the purification yield is calculated as the purified scFv per liter of refolding volume.
  • the assessment of the conformational stability, monodispersity and structural integrity of the scFv constructs is an integral component for the ranking of the different molecules with respect to the developability.
  • a prerequisite for the meaningful comparison of the different constructs is the preparation of purified molecules of similar quality.
  • the criterion "monomer purity" determined by SE-HPLC is intended to ensure compatible quality of the different test substances.
  • SDS-PAGE for the determination of protein purity and identity was performed to confirm comparable quality of the tested preparations.
  • the thermal unfolding behavior of the lead candidates was tested by differential scanning fluorimetry (DSF) to allow ranking of the molecules with respect to their expected conformational stability.
  • DSF differential scanning fluorimetry
  • a normalized plot of the fluorescence raw data is shown in Figure 3, which depicts duplicate measurements of each sample.
  • a cooperative unfolding behavior was observed.
  • the two molecules 16-22-H05-sc02 and 16-22-H05-sc04 showed a T m of 71.3 and 72.6°C, respectively.
  • High domain stability indicates dense packaging of amino acid residues, which also correlates with resistance towards protease cleavage.
  • the SE-HPLC assessment quantitatively determines the content of the monomeric fraction as well as of soluble oligomers/aggregates. Such soluble oligomers are oftentimes reversible and relatively loose associations driven by electrostatic or hydrophobic interactions between correctly folded proteins.
  • Tm As assessed by thermal unfolding and the propensity for oligomer/aggregate formation as assessed by SE-HPLC particularly for proteins with "border line” stability. Beyond a certain threshold Tm of approximately 60°C antibody variable domains are generally sufficiently stable to be resistant toward aggregation/precipitation and proteolytic degradation due to partial domain unfolding at ambient temperature.
  • Oligomerization driven by hydrophobic and/or electrostatic interactions of surface residues may, however, still occur.
  • elevated temperature e.g. 37°C
  • the various mechanisms of oligomer formation and precipitation may occur simultaneously.
  • Binding kinetics (ka, kd and KD) to human TNFa and potency to neutralize TNFoinduced apoptosis of L929 fibroblasts was analyzed. Additionally, the potency to inhibit Cynomolgus monkey (Macaca fascicularis) and Rhesus monkey (Macaca mulatta) TNFa induced apoptosis as well as the potency to inhibit the interaction between human TNFa and TNFRI/TNFRII by ELISA and target selectivity for binding to TNFa over TNFp was determined.
  • Affinity of humanized scFvs to human TNFa was determined by SPR measurements (see also 2.1.1 ). Affinity was determined using 2-fold serial dilutions of the respective scFvs.
  • the scFvs were derived from a rabbit monoclonal antibody. Two scFv variants were generated , named "CDR" (CDR) and "structural graft” (STR).
  • CDR CDR
  • STR structural graft
  • the ability of the humanized scFvs to neutralize human TNFa was analyzed using the L929 assay (see also 2.1 .2).
  • the potency (IC 5 o and IC 9 o) to neutralize TNFa induced apoptosis was analyzed for 16-22-H5 derived scFvs and compared to the potency of the reference antibody infliximab to allow for direct comparison of IC 50 and IC 90 values from different assay plates.
  • Relative IC 50 and IC 90 values were calculated in mass units (ng/ml) of infliximab and the scFvs. Potency analysis was performed several times on different days with different lots of antibody fragments.
  • Figure 7 shows representative dose-response curves from one experiment for each of the two scFvs. Mean values of replicate measurements are shown in Table 7 (standard deviations are summarized in the legend of the table).
  • the humanized scFvs inhibited TNFa induced apoptosis with lower IC 50 and IC 90 values than infliximab (see Table 7). Consistent with SPR results, the domain shuffled variant 16- 22-H05-sc04 (CDR/STR) exhibited equal potency when compared to the structural graft 16- 22-H05-sc02 (STR).
  • the scFvs 16-22-H05-sc04 and 16-22-H05-sc02 showed excellent TNFa-neutralizing activities, with IC 50 values of 14.6- and 13.1 -fold better than infliximab, respectively.
  • IC 90 values of 16-22-H05-sc04 and 16-22-H05-sc02 were 13.1 - and 12.6-fold better than for infliximab, respectively.
  • Species cross-reactivity for the top ranking scFvs was determined by two methods: 1 ) potency to neutralize Cynomolgus monkey and Rhesus monkey TNFa in the L929 assay and 2) affinity to Cynomolgus monkey and Rhesus monkey TNFa by SPR.
  • the potency to neutralize TNFa from the different species was determined by the L929 assay similarly as described above for human TNFa using Cynomolgus monkey and Rhesus monkey TNFa, respectively (see also 2.1 .2). TNFa from both species showed very similar potency to induce L929 apoptosis (data not shown). Therefore, same concentrations of human and monkey TNFa were used for species cross-reactivity testing. Additionally, binding kinetics (by SPR) to Cynomolgus monkey and Rhesus monkey TNFa were determined using a similar assay as for human TNFa (see also 2.1.1 ).
  • All scFvs derived from the clone 16-22-H05 showed cross-reactivity to Cynomolgus monkey and Rhesus monkey TNFa (see Table 7).
  • the affinities were similar, namely 2.0 x 10 "10 and 2.3 x 10 "10 M for Cynomolgus monkey and Rhesus monkey, respectively.
  • the difference in affinity between human and monkey TNFa was about 5-fold. Potencies to neutralize Cynomolgus monkey, Rhesus monkey and human TNFa correlated well with the affinities to the respective TNFas. Consequently the two clones derived from 16-22-H05 showed between 5- to 7-fold lower potencies towards monkey TNFa as compared to human TNFa (see Table 7 and Figure 8). To summarize, the two scFvs showed species cross-reactivity to Cynomolgus and Rhesus TNFa.
  • the potency of each humanized scFv to inhibit the interaction between human TNFa and TNFRI/II was assessed by ELISA (see 2.1.3).
  • individual IC 50 values on each plate were calibrated against the IC 50 of the reference molecule infliximab that was taken along on each plate and relative IC 50 and IC 90 values were calculated in mass units (ng/ml) of Infliximab and the scFvs.
  • Neutralization assays can distinguish potencies of target blocking antibodies only if they bind their target with an equilibrium binding constant (KD) that is higher than the target concentration used in the potency assay (KD > target concentration).
  • the L929 assay can differentiate potencies between scFvs with KD > 5 pM, while the inhibition ELISA can only differentiate potencies between scFvs with KD > 960 pM. Since all of the scFvs analyzed showed KDs below 960 pM, potencies between scFvs with different affinities (but similar mechanism of action) can be differentiated only in the L929 assay.
  • 16-22-H5-sc02 and 16-22-05-sc04 showed potencies for blocking of the TNFa-TNFRI interaction between 2.8 and 3.5-fold higher compared to infliximab while the potency compared to infliximab in the L929 assay was significantly higher (13.1 and 14.6-fold).
  • potencies of the scFvs are in general slightly higher compared to the parental IgG although affinities in general are in the same range for the parental rabbit IgG.
  • results from the inhibition ELISA were not used for ranking of potencies between the different antibodies but primarily for comparison of the potential of antibodies to block the interaction with TNFRI versus TNFRII.
  • the investigated scFvs blocked the interaction between both TNFa receptors with comparable potencies (Table 9, Figure 9 and Figure 10).
  • the selectivity for binding to TNFa over TNFp is significantly higher than approximately 5000 to 20 ⁇ 00 fold for all of the fragments tested (see also Table 7). Therefore, off-target binding of any of the scFvs appears highly unlikely.
  • Binding affinities of scFvs towards human TNFa were measured by surface plasmon resonance (SPR) using a MASS-1 SPR instrument (Sierra Sensors). Performance of the SPR assay was qualified by analysis of a reference antibody antigen interaction such as certolizumab-TNFa interaction. The pegylated Fab-fragment certolizumab was selected as reference due to its monovalent binding mode similar to that of scFvs. Using the same assay setup as for affinity measurements of the scFvs, a value of 9.94 x 10 "11 M was determined for the affinity of certolizumab to TNFa. This value is in good agreement with published K D values of 9.02 ⁇ 1.43 x 10 "11 M (BLA certolizumab; BLA number: 125160; submission date: April 30, 2007).
  • scFvs human TNFa (Peprotech, Cat. No. 300-01 ) was immobilized on a sensor chip (SPR-2 Affinity Sensor, Amine, Sierra Sensors) by amine- coupling to reach an immobilization level of 50 to 100 RUs (immobilization levels achieved during SPR analysis were between 40 to 120 RUs).
  • affinity screening of scFvs was performed using only one scFv concentration (90 nM).
  • SiCK Single Injection Cycle Kinetics
  • L929 fibroblasts ATCC/LGC Standards, Cat. No. CCL-1 .
  • L929 cells were sensitized to TNFa-induced apoptosis by addition of 1 ⁇ g ml actinomycin D.
  • the used TNFa concentration (5 pM) induces submaximal L929 apoptosis (EC 90 ).
  • the cells were incubated for 24 hours. Survival of the cells was determined by a colorimetric assay using the WST-8 (2- (2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, mono- sodium salt) cell proliferation reagent (Sigma Aldrich, Cat. No. 96992).
  • WST-8 is reduced by cellular dehydrogenases to an orange formazan product. The amount of formazan produced is directly proportional to the number of living cells.
  • TNFa Species cross-reactivity to Cynomolgus monkey (Sino Biological, Cat. No. 90018-CNAE) and Rhesus monkey (R&D Systems, Cat. No. 1070-RM-025/CF) TNFa was measured using the same assay setup and applying the same quality measures as described above for human TNFa. Similarly to the human counterpart, TNFa concentrations that induce submaximal L929 apoptosis (EC 90 ) were used for species cross-reactivity testing. TNFa from both species showed very similar potency to human TNFa to induce L929 mouse fibroblast apoptosis. Consequently the same concentration of TNFa (5 pM) was used for oth species tested. During species cross-reactivity testing CVs of most of the duplicate measurement points were below 10%.
  • the inhibitory effect of scFvs on ligand binding was assessed using an ELISA, a biochemical method solely reproducing the interaction between TNFa and TNFRI and TNFRII.
  • the extracellular domain of TNFRI fused to the Fc region of human IgG was coated on a 96-well Maxisorp ELISA at a concentration of 0.5 ⁇ g ml.
  • the extracellular domain of TNFRII fused to the Fc region of human IgG was coated at a concentration of 2 ⁇ g ml. All subsequent steps were identical for both assays. In order to detect binding of TNFa to TNFRI and TNFRII, TNFa was biotinylated prior to its use.
  • Biotinylated human TNFa (960 pM, 50 ng/ml) was first incubated with 3-fold serially diluted humanized anti-TNFa scFvs and infliximab (10 ⁇ 00 ng/ml-0.2 ng/ml) for 1 hour at room temperature.
  • the TNFa/antibody fragment mixtures were transferred to the TNF receptor immobilized plates and binding of unblocked TNFa to the immobilized TNFa receptor was detected after incubation at room temperature for 20 minutes with the biotin-binding streptavidin-HRP (SDT Reagents, Cat. No. SP40C).
  • TMB 3',5,5'-tetramethylbenzidine
  • TNFa binding to the most homologous family member TNFp was assessed.
  • the potential to inhibit the interaction of biotinylated TNFa with scFvs by unlabeled TNFp (Peprotech, Cat. No. 300-01 B) and TNFa (Peprotech, Cat. No. 300-01 ) was analyzed by competition ELISA.
  • the scFvs were coated on a 96-well Maxisorp ELISA plate at a concentration of 1 ⁇ g ml.
  • TNFp did not show any significant inhibition of the interaction between biotinylated TNFa and scFvs (see also Figure 1 1 ).
  • the IC 50 to inhibit the interaction by TNFp relative to TNFa was calculated.
  • the selectivity for binding to TNFa over TNFp was determined to be significantly higher than 5 ⁇ 00 to 20'000-fold.
  • the dose- response curves were analyzed in duplicate. Standard deviations and CVs were calculated for each measurement point (CV ⁇ 25% for all but one of the TNFa/ ⁇ concentrations tested). All scFv fulfilled this criterion.
  • SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
  • USP United States Pharmacopeia
  • analytical gel electrophoresis is an appropriate and routine method to identify and to assess the homogeneity of proteins in drug substances. The method is used to quantify the amount of scFv product from E.coli lysates to derive the expression yield after fermentation. Another application of the method is to verify the identity of test substances based on their molecular weight with respect to the theoretical values. For supportive purposes this method is used to quantify the purity of test samples with respect to process-related impurities (host cell proteins) and product related impurities (degradation products or adducts).
  • SDS-PAGE allows for specific detection of the protein of interest in the mixture of host cell proteins.
  • a reference standard dilution series in the linear range of the method (which was determined in advance) was included on every gel.
  • a linear regression of band intensities (measured by densitometry) versus nominal concentrations of the reference standard were used to calculate a standard curve, which in turn, was used to extrapolate scFv content in the sample.
  • the lysate samples of unknown product concentrations were loaded in different dilutions (at least 1 :10 in dilution buffer) to have at least one scFv concentration in the linear range of the method.
  • the product amount was calculated based on the measured band intensities of the scFv and the concentration was determined using the dilution factors of the sample preparation. The values were averaged for all samples that were within the linear range of the standard curve.
  • an inhibition/enhancement test was performed by spiking a lysate sample with a known amount of reference standard.
  • the lower limit of detection (LOD) for a reference scFv was determined visually (by identifying the protein band) at a nominal load of 0.02 ⁇ g, the evaluation of the intensity histogram of the respective lane shows a signal-to-noise ratio at this load of approximately 2.
  • the linear range for the quantification was determined by analyzing the main bands densitometrically. The fit of the data with a linear regression, results in a coefficient of determination (R 2 ) of 0.9998, thus indicating a good quality of the fit.
  • R 2 coefficient of determination
  • the relative error of each individual data point was determined to document the suitability of the method in the chosen range. The relative errors are below 10% for all data points indicating good accuracy of this method.
  • UV absorbance at 280 nm is a total protein assay as outlined in USP Chapter 1057. Protein solutions absorb UV light at a wavelength of 280 nm due to the presence of aromatic amino acids. The UV absorbance is a function of the content of tyrosine and tryptophan residues in the protein and is proportional to the protein concentration.
  • the measurement of the UV absorbance was performed with the Infinity reader M200 Pro equipped with Nanoquant plate (Tecan Group Ltd.).
  • the absorbance of the protein samples were measured at 280 nm and 310 nm, where the latter wavelength was serving as a reference signal that was subtracted from the 280 nm signal.
  • a blank subtraction was performed for each measurement.
  • the final absorbance signal of a protein sample obtained was used to calculate the protein concentration using Lambert-Beer's law.
  • SE-HPLC is a separation technique based on a solid stationary phase and a liquid mobile phase as outlined by the USP chapter 621 . This method separates molecules based on their size and shape utilizing a hydrophobic stationary phase and aqueous mobile phase. The separation of molecules is occurring between the void volume (V 0 ) and the total permeation volume (V T ) of a specific column. Measurements by SE-HPLC were performed on a Chromaster HPLC system (Hitachi High-Technologies Corporation) equipped with automated sample injection and a UV detector set to the detection wavelength of 280 nm. The equipment is controlled by the software EZChrom Elite (Agilent Technologies, Version 3.3.2 SP2) which also supports analysis of resulting chromatograms.
  • Protein samples were cleared by centrifugation and kept at a temperature of 6°C in the autosampler prior to injection.
  • the column Shodex KW402.5-4F Showa Denko Inc., #F6989201
  • a standardized buffered saline mobile phase 50 mM Sodium acetate pH 6.0, 250 mM sodium chloride
  • the target sample load per injection was 5 ⁇ g.
  • Samples were detected by an UV detector at a wavelength of 280 nm and the data recorded by a suitable software suite. The resulting chromatograms were analyzed in the range of V 0 to V T thereby excluding matrix associated peaks with >10 min elution time.
  • a reference standard was routinely measured at the beginning and end of each HPLC sequence.
  • the reference standard used for this system suitability test was a scFv that had been produced as a batch and was aliquoted to be used for each measurement timepoint.
  • the method DSF is a non-compendial method to measure temperature-dependent protein unfolding.
  • the measurement of the thermal unfolding temperature by DSF were performed with a MX3005P qPCR machine (Agilent Technologies) controlled with the MX Pro software package (Agilent Technologies) and equipped with an excitation/emission filter set at 492/610 nm.
  • the reactions were set-up in Thermo fast 96 white PCR plates (Abgene; #AB- 0600/W).
  • a commercially available stock solution of the dye SYPRO orange (Molecular Probes; # S6650) was used at a final dilution of 1 :1 ⁇ 00.
  • the protein samples were diluted for the unfolding measurements to a final concentration of 50 ⁇ g/mL in a standardized buffered saline solution.
  • the thermal unfolding was performed by a temperature program starting at 25°C ramping up to 96°C in 1 °C steps with a duration of 30 seconds. During the temperature program the fluorescence emission of each sample was recorded.
  • the recorded raw data was processed and evaluated with a package of Microsoft Excel templates (Niesen, Nature Protocols 2007, Vol. 2 No.9) and the fluorescence data was fitted with a Boltzmann equation using the program GraphPad Prism (GraphPad Software, Inc.) to obtain the midpoint of transition (T m ).
  • the single-chain diabody construct was designed by arranging the variable domains in a VLA-L1 -VHB-L2-VLB-L3-VHA configuration. In these constructs the VLA and VHA and VLB and VHB domains jointly form the binding site for TNFa.
  • the peptide linkers L1 -L3 connecting the variable domains were constructed of glycine/serine repeats.
  • the two short linkers L1 and L3 are composed of a single G 4 S repeat, whereas the long linker L2 is composed of the sequence (G 4 S) 4 .
  • Example 2 The nucleotide sequences encoding the humanized variable domains (Example 2; 1.2.1.) were de novo synthesized and cloned into an adapted vector for E.coli expression that is based on a pET26b(+) backbone (Novagen). The expression and purification was performed as described for the scFvs in Example 2; 1 .2.1.
  • the humanized IgG was constructed by cloning the variable domains a suitable mammalian expression vector for transient heterologous expression containing a leader sequence and the respective constant domains e.g. the pFUSE-rlgG vectors (Invivogen).
  • the transient expression of the functional IgG was performed by co-transfection of vectors encoding the heavy and light chains with the FreeStyleTM MAX system in CHO S cells. After cultivation for several days the supernatant of the antibody secreting cells was recovered for purification. Subsequently the secreted IgGs were affinity purified by Protein A sepharose (GE Healthcare). The elution fractions were analyzed by SDS-PAGE, UV absorbance at 280 nm and SE-HPLC.
  • the affinities of the antibody molecules were determined using a Biacore instrument as described in Example 2 under 2.1 .1 ).
  • 16-22-H5- scFv and TNFa were incubated at two different molar ratios, namely at a 1 :1 and 4.5:1 molar ratio. Since TNFa exists as a trimer in solution the indicated molar ratios refer to the TNFatrimer- Thus, in the 4.5:1 ratio the 16-22-H5-scFv is in excess and should occupy all TNFatrimer binding positions resulting in complexes of 1 TNFa tr imer with 3 scFv.
  • TNFa and 16-22-H5-scFv were incubated for 2 hours at RT to allow for complex formation. Samples were then centrifuged at 4°C for 10 min. 10 ⁇ _ of each sample were analysed on SE-HPLC.
  • the SE-HPLC analysis was performed with 50 mM phosphate buffer pH 6.5, 300 mM NaCI as eluent at a flow rate of 0.35 mL/min. Eluted protein peaks were detected at a wavelength of 280 nm.
  • the column was calibrated using the Gel filtration Calibration Kit from GE Healthcare (LMW, HMW) in advance for the determination of apparent molecular weights.
  • the bottom panel of Figure 12 shows the elution profile with equimolar amounts of scFv and TNFa which is overlayed with the profiles of TNFa tr imer alone and scFv alone. Due to the trimerization of the TNFa in solution there are theoretically up to three equivalent binding sites for the scFv present on each trimer and hence the scFv molecules are limiting. Under these conditions all three complex species (3:1 , 2:1 , 1 :1 ) were identified. The top panel of figure 12 shows the elution profile of the complex with excess amounts of scFv. The surplus of unbound scFv eluted at the expected retention time. The TNFa peak was quantitatively consumed for complex formation and disappeared completely.
  • the apparent binding stoichiometry was also calculated based on the apparent MW of the 16-22-H5-scFv:TNFa complex as determined by SE-HPLC. Based on retention time, the apparent MW was calculated to be 139.7 kDa. According to equation (1 ) below the apparent binding stoichiometry was calculated to be 3.3. This correlates well with the theoretical number of three equivalent binding sites available for scFv on the TNFa tr imer and the observations above where a 3:1 binding stoichiometry was determined.
  • Figure 13 illustrates that the bivalent 16-22-H5-scDb is able to bind two TNFa molecules simultaneously while, as expected, the monovalent 16-22-H5-scFv binds only to one TNFa molecule. Further, the formation of TN Fa-antibody complexes was assessed at different ratios of TNFa and 16-22-H5 antibody formats using SE-HPLC. 16-22-H5-lgG (150 kDa) and 16-22-H5- scDb (52 kDa) were incubated with TNFa (52 kDa) at different molar ratios (1 :3, 1 :1 , 3:1 ) in respect to binding sites.
  • IgG and scDb have 2 and TNFa has 3 binding sites.
  • the antibody-TNFa mixtures were incubated for at least 30 min at 37°C, cooled down for 10 min at RT and stored overnight at 2 - 8°C.
  • Five to 10 uL of protein mixture at a concentration of approx. 1 mg/mL were injected onto a TOSHO TSKgel UP-SW3000 column.
  • the analysis was performed with 150 mM phosphate buffer pH 6.8, 100 mM NaCI as eluent at a flow rate of 0.3 mL/min. Eluted protein peaks were detected at a wavelength of 214 nm.
  • Figure 14A shows the formation of 16-22-H5-lgG:TNFa complexes. Complexes that are ⁇ 600 kDa indicate the formation of complexes consisting of ⁇ 2 TNFa and ⁇ 3 IgG molecules.
  • Figure 14B shows the formation of 16-22-H5-scDb:TNFa complexes. Complexes that are ⁇ 300 kDa indicate the formation of complexes consisting of ⁇ 2 TNFa and ⁇ 3 scDb molecules.
  • PBMC peripheral blood mononuclear cells
  • MLR mixed lymphocyte reaction
  • CD14 + monocytes in RPMI 1640 were seeded in 96-well plates and incubated for 16 h at 37°C/5 % C0 2 in a humidified incubator. Then cells were treated with anti-TNFa antibodies or IgG control antibody in duplicates for 1 h using final antibody concentrations ranging from 2 to 2000 ng/mL. The monocytes were washed 3 times with cell culture medium and subsequently incubated with LPS (100 ng/mL) for 4 h at 37°C/5% C0 2 . IL-1 ⁇ and TNFa concentrations in the cell culture supernatants were determined using commercially available ELISA kits (R&D Systems). The Results are shown in Tables 13 and 14 and Figures 16A and B.
  • IC 50 was determined using a four-parameter logistic curve fit. Regarding secretion of I L-1 ⁇ the ICso values for 16-22-H5-lgG, 16-22-H5-scDb, 16-22-H5-scFv and adalimumab is summarized in Table 13 below.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
PCT/EP2017/056237 2016-03-17 2017-03-16 Anti-tnfalpha-antibodies and functional fragments thereof WO2017158092A1 (en)

Priority Applications (35)

Application Number Priority Date Filing Date Title
SI201731446T SI3430043T1 (sl) 2016-03-17 2017-03-16 Anti-TNFalfa protitelesa in njihovi funkcionalni fragmenti
RS20231087A RS64830B1 (sr) 2016-03-17 2017-03-16 Anti-tnf-alfa antitela i njihovi funkcionalni fragmenti
CA3011784A CA3011784C (en) 2016-03-17 2017-03-16 Anti-tnf.alpha.-antibodies and functional fragments thereof
BR112018016667A BR112018016667A2 (pt) 2016-03-17 2017-03-16 anticorpos anti-tnf alfa e fragmentos funcionais dos mesmos
EP17710748.9A EP3430043B1 (de) 2016-03-17 2017-03-16 Anti-tnf-alpha-antikörper und funktionale fragmente davon
KR1020187022632A KR102470235B1 (ko) 2016-03-17 2017-03-16 항-TNFα-항체 및 이의 기능성 단편
PL17710748.9T PL3430043T3 (pl) 2016-03-17 2017-03-16 Przeciwciała anty-tnf-alfa i ich funkcjonalne fragmenty
HRP20231464TT HRP20231464T1 (hr) 2016-03-17 2017-03-16 Protutijela protiv tnf-alfa i njihovi funkcionalni fragmenti
MYPI2018001566A MY194041A (en) 2016-03-17 2017-03-16 Anti-tnfalpha-antibodies and functional fragments thereof
JP2019500013A JP7129398B2 (ja) 2016-03-17 2017-03-16 抗ＴＮＦα抗体およびそれらの機能的断片
DK17710748.9T DK3430043T5 (da) 2016-03-17 2017-03-16 Anti-tnfalpha-antistoffer og funktionelle fragmenter deraf
CN201780015298.5A CN109071650B (zh) 2016-03-17 2017-03-16 抗TNFα抗体及其功能片段
FIEP17710748.9T FI3430043T3 (fi) 2016-03-17 2017-03-16 Anti-TNFalfa -vasta-aineita ja niiden funktionaalisia fragmentteja
LTEPPCT/EP2017/056237T LT3430043T (lt) 2016-03-17 2017-03-16 Anti-tnf alfa antikūnai ir funkciniai jų fragmentai
CR20180448A CR20180448A (es) 2016-03-17 2017-03-16 ANTICUERPOS ANTI-TNFa Y FRAGMENTOS FUNCIONALES DE LOS MISMOS
US16/085,506 US11459383B2 (en) 2016-03-17 2017-03-16 Anti-TNF-alpha-antibodies and functional fragments thereof
EP23192170.1A EP4275745A3 (de) 2016-03-17 2017-03-16 Anti-tnf-alpha-antikörper und funktionale fragmente davon
MX2018011258A MX2018011258A (es) 2016-03-17 2017-03-16 Anticuerpos anti-tnf alfa y fragmentos funcionales de los mismos.
SG11201807062RA SG11201807062RA (en) 2016-03-17 2017-03-16 Anti-tnfalpha-antibodies and functional fragments thereof
KR1020227040533A KR20220158877A (ko) 2016-03-17 2017-03-16 항-TNFα-항체 및 이의 기능성 단편
ES17710748T ES2963221T3 (es) 2016-03-17 2017-03-16 Anticuerpos anti-TNFalfa y fragmentos funcionales de los mismos
MA43717A MA43717B1 (fr) 2016-03-17 2017-03-16 Anticorps anti-tnf alpha et fragments fonctionnels de ceux-ci
MDE20190099T MD3430043T2 (ro) 2016-03-17 2017-03-16 Anticorpi anti-TNF alfa și fragmente funcționale ale acestora
NZ745999A NZ745999A (en) 2016-03-17 2017-03-16 Anti-tnfalpha-antibodies and functional fragments thereof
TNP/2018/000298A TN2018000298A1 (en) 2016-03-17 2017-03-16 ANTl-TNFALPHA-ANTIBODIES AND FUNCTIONAL FRAGMENTS THEREOF.
EA201891788A EA039760B1 (ru) 2016-03-17 2017-03-16 Антитела к фно-альфа и их функциональные фрагменты
AU2017235387A AU2017235387B2 (en) 2016-03-17 2017-03-16 Anti-TNFalpha-antibodies and functional fragments thereof
IL261031A IL261031A (en) 2016-03-17 2018-08-07 Antibodies against tnf-alpha and functional fragments thereof
PH12018501907A PH12018501907A1 (en) 2016-03-17 2018-09-06 Anti-tnfalpha-antibodies and functional fragments thereof
SA518400027A SA518400027B1 (ar) 2016-03-17 2018-09-16 TNF- a أجسام مضادة لـ وشظايا وظيفية منها
CONC2018/0009783A CO2018009783A2 (es) 2016-03-17 2018-09-17 Anticuerpos-anti-tnfalfa-y fragmentos funcionales de los mismos
JP2022091271A JP2022130407A (ja) 2016-03-17 2022-06-06 抗ＴＮＦα抗体およびそれらの機能的断片
US17/890,857 US12043663B2 (en) 2016-03-17 2022-08-18 Anti-TNF-alpha-antibodies and functional fragments thereof
US17/958,759 US20230279091A1 (en) 2016-03-17 2022-10-03 Anti-tnf-alpha-antibodies and functional fragments thereof
AU2024219840A AU2024219840A1 (en) 2016-03-17 2024-09-18 Anti-tnfalpha-antibodies and functional fragments thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP16160907.8A EP3219726B1 (de) 2016-03-17 2016-03-17 Anti-tnf-alpha-antikörper und funktionale fragmente davon
EP16160907.8 2016-03-17

Related Child Applications (3)

Application Number Title Priority Date Filing Date
US16/085,506 A-371-Of-International US11459383B2 (en) 2016-03-17 2017-03-16 Anti-TNF-alpha-antibodies and functional fragments thereof
US17/890,857 Division US12043663B2 (en) 2016-03-17 2022-08-18 Anti-TNF-alpha-antibodies and functional fragments thereof
US17/958,759 Division US20230279091A1 (en) 2016-03-17 2022-10-03 Anti-tnf-alpha-antibodies and functional fragments thereof

Publications (1)

Publication Number Publication Date
WO2017158092A1 true WO2017158092A1 (en) 2017-09-21

Family

ID=55542576

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2017/056237 WO2017158092A1 (en) 2016-03-17 2017-03-16 Anti-tnfalpha-antibodies and functional fragments thereof

Country Status (40)

Country Link
US (3) US11459383B2 (de)
EP (3) EP3219726B1 (de)
JP (2) JP7129398B2 (de)
KR (2) KR102470235B1 (de)
CN (1) CN109071650B (de)
AR (1) AR107917A1 (de)
AU (2) AU2017235387B2 (de)
BR (1) BR112018016667A2 (de)
CA (1) CA3011784C (de)
CL (1) CL2018002649A1 (de)
CO (1) CO2018009783A2 (de)
CR (1) CR20180448A (de)
CY (1) CY1123784T1 (de)
DK (2) DK3219726T3 (de)
EA (1) EA039760B1 (de)
ES (2) ES2836349T3 (de)
FI (1) FI3430043T3 (de)
GE (1) GEP20217307B (de)
HR (2) HRP20231464T1 (de)
HU (2) HUE052773T2 (de)
IL (1) IL261031A (de)
JO (2) JOP20170064B1 (de)
LT (2) LT3219726T (de)
MA (1) MA43717B1 (de)
MD (1) MD3430043T2 (de)
MX (1) MX2018011258A (de)
MY (1) MY194041A (de)
NZ (1) NZ745999A (de)
PH (1) PH12018501907A1 (de)
PL (2) PL3219726T3 (de)
PT (2) PT3219726T (de)
RS (2) RS61374B1 (de)
SA (1) SA518400027B1 (de)
SG (1) SG11201807062RA (de)
SI (2) SI3219726T1 (de)
TN (1) TN2018000298A1 (de)
TW (2) TWI829444B (de)
UY (1) UY37157A (de)
WO (1) WO2017158092A1 (de)
ZA (4) ZA202108602B (de)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020115277A1 (en) 2018-12-07 2020-06-11 Tillotts Pharma Ag Topical treatment of immune checkpoint inhibitor induced diarrhoea, colitis or enterocolitis using antibodies and fragments thereof
US20200216527A1 (en) * 2017-09-19 2020-07-09 Tillotts Pharma Ag Antibody variants
US20200255511A1 (en) * 2017-09-19 2020-08-13 Tillotts Pharma Ag Antibody variants
US10759852B2 (en) 2016-03-17 2020-09-01 Numab Innovation Ag Anti-TNF-alpha-antibodies and functional fragments thereof
US10787508B2 (en) 2016-03-17 2020-09-29 Numab Innovation Ag Anti-TNFα-antibodies and functional fragments thereof
JP2020533996A (ja) * 2017-09-19 2020-11-26 ティロッツ ファーマ アーゲーＴｉｌｌｏｔｔｓ Ｐｈａｒｍａ Ａｇ 抗体バリアント
US10975143B2 (en) 2016-03-17 2021-04-13 Tillotts Pharma Ag Anti-TNF-α-antibodies and functional fragments thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SI3219726T1 (sl) * 2016-03-17 2021-02-26 Tillotts Pharma Ag Anti-TNF alfa protitelesa in njihovi funkcionalni fragmenti

Citations (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4444887A (en) 1979-12-10 1984-04-24 Sloan-Kettering Institute Process for making human antibody producing B-lymphocytes
WO1986001533A1 (en) 1984-09-03 1986-03-13 Celltech Limited Production of chimeric antibodies
EP0239400A2 (de) 1986-03-27 1987-09-30 Medical Research Council Rekombinante Antikörper und Verfahren zu deren Herstellung
US4716111A (en) 1982-08-11 1987-12-29 Trustees Of Boston University Process for producing human antibodies
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
EP0392745A2 (de) 1989-04-05 1990-10-17 Celltech Limited Immunokonjugate und Prodrogen und ihre gemeinschaftliche Anwendung für die Verabreichung von Arzneimitteln
WO1991009967A1 (en) 1989-12-21 1991-07-11 Celltech Limited Humanised antibodies
WO1991010741A1 (en) 1990-01-12 1991-07-25 Cell Genesys, Inc. Generation of xenogeneic antibodies
WO1992001047A1 (en) 1990-07-10 1992-01-23 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
WO1992011383A1 (en) 1990-12-21 1992-07-09 Celltech Limited RECOMBINANT ANTIBODIES SPECIFIC FOR TNF$g(a)
EP0519596A1 (de) 1991-05-17 1992-12-23 Merck & Co. Inc. Verfahren zur Verringerung der Immunogenität der variabelen Teile von Antikörpern
US5219996A (en) 1987-09-04 1993-06-15 Celltech Limited Recombinant antibodies and methods for their production in which surface residues are altered to cysteine residues for attachment of effector or receptor molecules
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
EP0592106A1 (de) 1992-09-09 1994-04-13 Immunogen Inc Oberflächenumformung von rodenten Antikörpern
US5413923A (en) 1989-07-25 1995-05-09 Cell Genesys, Inc. Homologous recombination for universal donor cells and chimeric mammalian hosts
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
GB2297145A (en) 1995-01-23 1996-07-24 Western Atlas Int Inc EBW perforating gun system
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
WO1996033735A1 (en) 1995-04-27 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1996034096A1 (en) 1995-04-28 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5658570A (en) 1991-07-25 1997-08-19 Idec Pharmaceuticals Corporation Recombinant antibodies for human therapy
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
WO1998016654A1 (en) 1996-10-11 1998-04-23 Japan Tobacco, Inc. Production of a multimeric protein by cell fusion method
WO1998024893A2 (en) 1996-12-03 1998-06-11 Abgenix, Inc. TRANSGENIC MAMMALS HAVING HUMAN IG LOCI INCLUDING PLURAL VH AND Vλ REGIONS AND ANTIBODIES PRODUCED THEREFROM
US5807715A (en) 1984-08-27 1998-09-15 The Board Of Trustees Of The Leland Stanford Junior University Methods and transformed mammalian lymphocyte cells for producing functional antigen-binding protein including chimeric immunoglobulin
US5814318A (en) 1990-08-29 1998-09-29 Genpharm International Inc. Transgenic non-human animals for producing heterologous antibodies
WO1998046645A2 (en) 1997-04-14 1998-10-22 Micromet Gesellschaft Für Biomedizinische Forschung Mbh Method for the production of antihuman antigen receptors and uses thereof
WO1998050433A2 (en) 1997-05-05 1998-11-12 Abgenix, Inc. Human monoclonal antibodies to epidermal growth factor receptor
US5885793A (en) 1991-12-02 1999-03-23 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
US5919452A (en) 1991-03-18 1999-07-06 New York University Methods of treating TNFα-mediated disease using chimeric anti-TNF antibodies
EP0948544A1 (de) 1996-12-10 1999-10-13 Celltech Therapeutics Limited Monovalente antikörperfragmente
WO2005117984A2 (en) 2004-06-01 2005-12-15 Celltech R & D Limited Albumin-binding conjugates comprising a fatty acid and peg
WO2006131013A2 (en) 2005-06-07 2006-12-14 Esbatech Ag STABLE AND SOLUBLE ANTIBODIES INHIBITING TNFα
US20070280931A1 (en) 1998-11-18 2007-12-06 Chen Yvonne M Antibody variants
WO2009155723A2 (en) 2008-06-25 2009-12-30 Esbatech, An Alcon Biomedical Research Unit Llc STABLE AND SOLUBLE ANTIBODIES INHIBITING TNFα
WO2012007880A2 (en) 2010-07-16 2012-01-19 Ablynx Nv Modified single domain antigen binding molecules and uses thereof
US8293235B2 (en) 2008-06-25 2012-10-23 ESBATech, an Alcon Biomedical Research Unit, LLC Humanization of rabbit antibodies using a universal antibody framework
EP2623515A1 (de) 2010-09-30 2013-08-07 Chengdu Kanghong Biotechnologies Co., Ltd. Humanisierte anti-tnf-antikörper und fragment-antigenbindung sowie ihre verwendung
US8697074B2 (en) 2008-07-10 2014-04-15 Esbatech, An Alcon Biomedical Research Unit Llc Methods and compositions for enhanced delivery of macromolecules
WO2014206561A1 (en) 2013-06-26 2014-12-31 Numab Ag Novel antibody frameworks
WO2015065987A1 (en) 2013-11-01 2015-05-07 Ibc Pharmaceuticals, Inc. Bispecific antibodies that neutralize both tnf-alpha and il-6: novel therapeutic agent for autoimmune disease
WO2015144852A1 (en) 2014-03-26 2015-10-01 Delenex Therapeutics Ag Binding members to tnf alpha

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103275221B (zh) * 1996-02-09 2016-08-17 艾伯维生物技术有限公司 结合人TNFα的人抗体
US7431927B2 (en) 2005-03-24 2008-10-07 Epitomics, Inc. TNFα-neutralizing antibodies
AU2011237679B2 (en) * 2010-04-07 2014-11-06 Abbvie Inc. TNF-alpha binding proteins
SI3219726T1 (sl) * 2016-03-17 2021-02-26 Tillotts Pharma Ag Anti-TNF alfa protitelesa in njihovi funkcionalni fragmenti
ES2843974T3 (es) 2016-03-17 2021-07-21 Tillotts Pharma Ag Anticuerpos anti-TNF alfa y fragmentos funcionales de los mismos
WO2017158079A1 (en) 2016-03-17 2017-09-21 Numab Innovation Ag Anti-tnfalpha-antibodies and functional fragments thereof
EP3430042B1 (de) 2016-03-17 2023-05-24 Numab Innovation AG Anti-tnf-alpha-antikörper und funktionale fragmente davon

Patent Citations (60)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4444887A (en) 1979-12-10 1984-04-24 Sloan-Kettering Institute Process for making human antibody producing B-lymphocytes
US4716111A (en) 1982-08-11 1987-12-29 Trustees Of Boston University Process for producing human antibodies
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5807715A (en) 1984-08-27 1998-09-15 The Board Of Trustees Of The Leland Stanford Junior University Methods and transformed mammalian lymphocyte cells for producing functional antigen-binding protein including chimeric immunoglobulin
WO1986001533A1 (en) 1984-09-03 1986-03-13 Celltech Limited Production of chimeric antibodies
EP0239400A2 (de) 1986-03-27 1987-09-30 Medical Research Council Rekombinante Antikörper und Verfahren zu deren Herstellung
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5219996A (en) 1987-09-04 1993-06-15 Celltech Limited Recombinant antibodies and methods for their production in which surface residues are altered to cysteine residues for attachment of effector or receptor molecules
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5693762A (en) 1988-12-28 1997-12-02 Protein Design Labs, Inc. Humanized immunoglobulins
US5693761A (en) 1988-12-28 1997-12-02 Protein Design Labs, Inc. Polynucleotides encoding improved humanized immunoglobulins
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US6180370B1 (en) 1988-12-28 2001-01-30 Protein Design Labs, Inc. Humanized immunoglobulins and methods of making the same
EP0392745A2 (de) 1989-04-05 1990-10-17 Celltech Limited Immunokonjugate und Prodrogen und ihre gemeinschaftliche Anwendung für die Verabreichung von Arzneimitteln
US5413923A (en) 1989-07-25 1995-05-09 Cell Genesys, Inc. Homologous recombination for universal donor cells and chimeric mammalian hosts
GB2246570A (en) 1989-12-21 1992-02-05 Celltech Ltd Humanised antibodies
WO1991009967A1 (en) 1989-12-21 1991-07-11 Celltech Limited Humanised antibodies
US5939598A (en) 1990-01-12 1999-08-17 Abgenix, Inc. Method of making transgenic mice lacking endogenous heavy chains
WO1991010741A1 (en) 1990-01-12 1991-07-25 Cell Genesys, Inc. Generation of xenogeneic antibodies
WO1992001047A1 (en) 1990-07-10 1992-01-23 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
US5814318A (en) 1990-08-29 1998-09-29 Genpharm International Inc. Transgenic non-human animals for producing heterologous antibodies
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US20030199679A1 (en) 1990-12-21 2003-10-23 Adair John Robert Recombinant antibodies specific for TNF-alpha
WO1992011383A1 (en) 1990-12-21 1992-07-09 Celltech Limited RECOMBINANT ANTIBODIES SPECIFIC FOR TNF$g(a)
US5919452A (en) 1991-03-18 1999-07-06 New York University Methods of treating TNFα-mediated disease using chimeric anti-TNF antibodies
EP0519596A1 (de) 1991-05-17 1992-12-23 Merck & Co. Inc. Verfahren zur Verringerung der Immunogenität der variabelen Teile von Antikörpern
US5681722A (en) 1991-07-25 1997-10-28 Idec Pharmaceuticals Corporation Recombinant antibodies for human therapy
US5693780A (en) 1991-07-25 1997-12-02 Idec Pharmaceuticals Corporation Recombinant antibodies for human therapy
US5658570A (en) 1991-07-25 1997-08-19 Idec Pharmaceuticals Corporation Recombinant antibodies for human therapy
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5885793A (en) 1991-12-02 1999-03-23 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
EP0592106A1 (de) 1992-09-09 1994-04-13 Immunogen Inc Oberflächenumformung von rodenten Antikörpern
GB2297145A (en) 1995-01-23 1996-07-24 Western Atlas Int Inc EBW perforating gun system
WO1996033735A1 (en) 1995-04-27 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1996034096A1 (en) 1995-04-28 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5916771A (en) 1996-10-11 1999-06-29 Abgenix, Inc. Production of a multimeric protein by cell fusion method
WO1998016654A1 (en) 1996-10-11 1998-04-23 Japan Tobacco, Inc. Production of a multimeric protein by cell fusion method
WO1998024893A2 (en) 1996-12-03 1998-06-11 Abgenix, Inc. TRANSGENIC MAMMALS HAVING HUMAN IG LOCI INCLUDING PLURAL VH AND Vλ REGIONS AND ANTIBODIES PRODUCED THEREFROM
EP0948544A1 (de) 1996-12-10 1999-10-13 Celltech Therapeutics Limited Monovalente antikörperfragmente
WO1998046645A2 (en) 1997-04-14 1998-10-22 Micromet Gesellschaft Für Biomedizinische Forschung Mbh Method for the production of antihuman antigen receptors and uses thereof
WO1998050433A2 (en) 1997-05-05 1998-11-12 Abgenix, Inc. Human monoclonal antibodies to epidermal growth factor receptor
US20070280931A1 (en) 1998-11-18 2007-12-06 Chen Yvonne M Antibody variants
WO2005117984A2 (en) 2004-06-01 2005-12-15 Celltech R & D Limited Albumin-binding conjugates comprising a fatty acid and peg
WO2006131013A2 (en) 2005-06-07 2006-12-14 Esbatech Ag STABLE AND SOLUBLE ANTIBODIES INHIBITING TNFα
EP2390267B1 (de) 2005-06-07 2013-06-05 ESBATech - a Novartis Company LLC Stabile und lösliche Antikörper zur Hemmung von TNF(alpha)
WO2009155723A2 (en) 2008-06-25 2009-12-30 Esbatech, An Alcon Biomedical Research Unit Llc STABLE AND SOLUBLE ANTIBODIES INHIBITING TNFα
US8293235B2 (en) 2008-06-25 2012-10-23 ESBATech, an Alcon Biomedical Research Unit, LLC Humanization of rabbit antibodies using a universal antibody framework
US20140193400A1 (en) 2008-06-25 2014-07-10 ESBA Tech, an Alcon Biomedical Research Unit LLC Stable and soluble antibodies inhibiting tnf alpha
US8673310B2 (en) 2008-06-25 2014-03-18 ESBA Tech, an Alcon Biomedical Research Unit LLC Stable and soluble antibodies inhibiting TNFα
US8697074B2 (en) 2008-07-10 2014-04-15 Esbatech, An Alcon Biomedical Research Unit Llc Methods and compositions for enhanced delivery of macromolecules
WO2012007880A2 (en) 2010-07-16 2012-01-19 Ablynx Nv Modified single domain antigen binding molecules and uses thereof
EP2623515A1 (de) 2010-09-30 2013-08-07 Chengdu Kanghong Biotechnologies Co., Ltd. Humanisierte anti-tnf-antikörper und fragment-antigenbindung sowie ihre verwendung
WO2014206561A1 (en) 2013-06-26 2014-12-31 Numab Ag Novel antibody frameworks
WO2015065987A1 (en) 2013-11-01 2015-05-07 Ibc Pharmaceuticals, Inc. Bispecific antibodies that neutralize both tnf-alpha and il-6: novel therapeutic agent for autoimmune disease
WO2015144852A1 (en) 2014-03-26 2015-10-01 Delenex Therapeutics Ag Binding members to tnf alpha

Non-Patent Citations (55)

* Cited by examiner, † Cited by third party
Title
"Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, GROVE PUBLISHERS
"CDP 571: anti-TNF monoclonal antibody, BAY 103356, BAY W 3356, Humicade", DRUGS IN R & D, ADIS INTERNATIONAL, AUCKLAND, NZ, vol. 4, no. 3, 1 January 2003 (2003-01-01), pages 174 - 178, XP009176085, ISSN: 1174-5886 *
"Poly(ethyleneglycol) Chemistry and Biological Applications", 1997, AMERICAN CHEMICAL SOCIETY
"Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, PLENUM PRESS
"Remington's Pharmaceutical Sciences", 1980
ADORINI ET AL., TRENDS IN IMMUNOLOGY TODAY, vol. 18, 1997, pages 209 - 211
BEUTLER ET AL., SCIENCE, vol. 234, 1985, pages 470 - 474
BODMER ET AL., CRITICAL CARE MEDICINE, vol. 21, 1993, pages S441 - S446
BORRAS ET AL., JBC, vol. 285, 2010, pages 9054 - 9066
BORRAS ET AL., JBC., vol. 285, 2010, pages 9054 - 9066
CHAPMAN, ADVANCED DRUG DELIVERY REVIEWS, vol. 54, 2002, pages 531 - 545
COPPIETERS ET AL., ARTHRITIS & RHEUMATISM, vol. 54, 2006, pages 1856 - 1866
DUBOWCHIK ET AL., PHARMACOLOGY AND THERAPEUTICS, vol. 83, 1999, pages 67 - 123
FELDMAN ET AL., ADVANCES IN IMMUNOLOGY, vol. 64, 1997, pages 283 - 350
FELDMAN ET AL., TRANSPLANTATION PROCEEDINGS, vol. 30, 1998, pages 4126 - 4127
FENDLY ET AL., HYBRIDOMA, vol. 6, 1987, pages 359 - 370
GERSHONI JONATHAN M ET AL: "Epitope mapping - The first step in developing epitope-based vaccines", BIODRUGS, ADIS INTERNATIONAL LTD, NZ, vol. 21, no. 3, 1 January 2007 (2007-01-01), pages 145 - 156, XP009103541, ISSN: 1173-8804, DOI: 10.2165/00063030-200721030-00002 *
GILLIES ET AL., J. IMMUNOL. METHODS, vol. 125, 1985, pages 191 - 202
HARLOW; LANE: "Antibodies, A Laboratory Manual", 1988, CSH PRESS
HARTMUT KUPPER ET AL: "Adalimumab (Humira)", 1 January 2007 (2007-01-01), XP002720017, ISBN: 978-3-527-31453-9, Retrieved from the Internet <URL:http://onlinelibrary.wiley.com/doi/10.1002/9783527619740.ch27/pdf> [retrieved on 20140210] *
HELLSTROM ET AL.: "Controlled Drag Delivery", 1987, pages: 623 - 53
JESPERS ET AL., BIOTECHNOLOGY, vol. 12, 1988, pages 899 - 903
KABAT E. A ET AL.: "Sequences of proteins of immunological interest", 1991, NIH PUBLICATION 91-3242
KIRCHNER ET AL., CYTOKINE, vol. 28, 2004, pages 67
KIRSCHENBAUM ET AL., CRITICAL CARE MEDICINE, vol. 26, 1998, pages 1625 - 1626
KUPPER H ET AL.: "Handbook of Therapeutic Antibodies", 1 January 2007, WILEY-VCH, article "Adalimumab (Humira", pages: 697 - 732
LALOR ET AL., EUR J IMMUNOL., vol. 22, 1992, pages 3001 - 2011
MARIA WIEKOWSKI ET AL: "Infliximab (Remicade)", 1 January 2007 (2007-01-01), XP002720018, ISBN: 978-3-527-31453-9, Retrieved from the Internet <URL:http://onlinelibrary.wiley.com/doi/10.1002/9783527619740.ch34/pdf> [retrieved on 20140210] *
MAZUMDAR S ET AL.: "mAbs", vol. 1, 1 September 2009, LANDES BIOSCIENCE, article "Golimumab", pages: 422 - 431
MAZUMDAR SOHINI ET AL: "Golimumab", MABS, LANDES BIOSCIENCE, US, vol. 1, no. 5, 1 September 2009 (2009-09-01), pages 422 - 431, XP002631538, ISSN: 1942-0870 *
MEAGER ET AL., HYBRIDOMA, vol. 6, 1987, pages 305 - 311
MELMED G Y ET AL.: "Nature Reviews. Drug Discovery", vol. 7, 1 August 2008, NATURE PUBLISHING GROUP, article "Certolizumab pegol", pages: 641 - 642
MELMED GIL Y ET AL: "Certolizumab pegol", NATURE REVIEWS. DRUG DISCOVERY, NATURE PUBLISHING GROUP, GB, vol. 7, no. 8, 1 August 2008 (2008-08-01), pages 641 - 642, XP009147828, ISSN: 1474-1784, DOI: 10.1038/NRD2654 *
MORRISON, SCIENCE, vol. 229, no. 4719, 1985, pages 1202 - 7
NIESEN, NATURE PROTOCOLS, vol. 2, no. 9, 2007
OI ET AL., BIOTECHNIQUES, vol. 4, 1986, pages 214 - 221
PADLAN, MOL. IMMUNOL, vol. 28, 1991, pages 489 - 498
RANKIN ET AL., BRITISH J. RHEUMATOLOGY, vol. 34, 1995, pages 334 - 342
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 7
ROGUSKA ET AL., PROC. NATL. ACAD. SCI., vol. 91, 1994, pages 969 - 973
RUDIKOFF S ET AL: "Single amino acid substitution altering antigen-binding specificity", PROCEEDINGS NATIONAL ACADEMY OF SCIENCES PNAS, NATIONAL ACADEMY OF SCIENCES, US, vol. 79, no. 6, 1 March 1982 (1982-03-01), pages 1979 - 1983, XP002683593, ISSN: 0027-8424, DOI: 10.1073/PNAS.79.6.1979 *
RUGS IN R&D, vol. 4, no. 3, 2003, pages 174 - 178
SALDANHA J W ET AL.: "Handbook of Therapeutic Antibodies", 1 January 2007, WILEY-VCH, article "Molecular Engineering I: Humanization", pages: 119 - 144
STEPHAN DUEBEL ED - STEFAN DÜBEL: "Handbook of Therapeutic Antibodies Chapter 6", 1 January 2007, HANDBOOK OF THERAPEUTIC ANTIBODIES, WILEY-VCH, WEINHEIM, PAGE(S) 119 - 144, ISBN: 978-3-527-31453-9, XP007913671 *
STEPHENS ET AL., IMMUNOLOGY, vol. 85, 1995, pages 668 - 674
STUDNICKA ET AL., PROT. ENG., vol. 7, 1994, pages 805 - 814
THORPE ET AL., IMMUNOL. REV., vol. 62, 1982, pages 119 - 58
TRACEY ET AL: "Tumor necrosis factor antagonist mechanisms of action: A comprehensive review", PHARMACOLOGY AND THERAPEUTICS, ELSEVIER, GB, vol. 117, no. 2, 26 October 2007 (2007-10-26), pages 244 - 279, XP022432143, ISSN: 0163-7258, DOI: 10.1016/J.PHARMTHERA.2007.10.001 *
URECH ET AL., ANN RHEUM DIS, vol. 69, 2010, pages 443
VOS ET AL., GASTROENTEROLOGY, vol. 140, 2011, pages 221
WALLIS, LANCET INFECT DIS, vol. 8, 2008, pages 601
WEIR ET AL., THERAPY, vol. 3, 2006, pages 535
WHERRY ET AL., CRITICAL CARE MEDICINE, vol. 21, 1993, pages S436 - S440
WIEKOWSKI M ET AL.: "Handbook of Therapeutic Antibodies", 1 January 2007, WILEY-VCH, article "Infliximab (Remicade", pages: 885 - 904
WILLIAMS ET AL., PNAS-USA, vol. 89, 1992, pages 9784 - 9788

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10759852B2 (en) 2016-03-17 2020-09-01 Numab Innovation Ag Anti-TNF-alpha-antibodies and functional fragments thereof
US10975143B2 (en) 2016-03-17 2021-04-13 Tillotts Pharma Ag Anti-TNF-α-antibodies and functional fragments thereof
US10787508B2 (en) 2016-03-17 2020-09-29 Numab Innovation Ag Anti-TNFα-antibodies and functional fragments thereof
JP2020534000A (ja) * 2017-09-19 2020-11-26 ティロッツ ファーマ アーゲーＴｉｌｌｏｔｔｓ Ｐｈａｒｍａ Ａｇ 抗体バリアント
US20200255511A1 (en) * 2017-09-19 2020-08-13 Tillotts Pharma Ag Antibody variants
US20200216527A1 (en) * 2017-09-19 2020-07-09 Tillotts Pharma Ag Antibody variants
JP2020533996A (ja) * 2017-09-19 2020-11-26 ティロッツ ファーマ アーゲーＴｉｌｌｏｔｔｓ Ｐｈａｒｍａ Ａｇ 抗体バリアント
JP7341127B2 (ja) 2017-09-19 2023-09-08 ティロッツ ファーマ アーゲー 抗体バリアント
JP7370965B2 (ja) 2017-09-19 2023-10-30 ティロッツ ファーマ アーゲー 抗体バリアント
US11999781B2 (en) * 2017-09-19 2024-06-04 Tillotts Pharma Ag Antibody variants
EP3456738B1 (de) * 2017-09-19 2024-07-17 Tillotts Pharma Ag Antikörpervarianten
WO2020115277A1 (en) 2018-12-07 2020-06-11 Tillotts Pharma Ag Topical treatment of immune checkpoint inhibitor induced diarrhoea, colitis or enterocolitis using antibodies and fragments thereof
WO2020114616A1 (en) 2018-12-07 2020-06-11 Tillotts Pharma Ag Topical treatment of immune checkpoint inhibitor induced diarrhoea, colitis or enterocolitis using antibodies and fragments thereof

Also Published As

Publication number Publication date
CN109071650A (zh) 2018-12-21
PL3430043T3 (pl) 2024-03-11
EP3430043B1 (de) 2023-08-30
DK3430043T5 (da) 2024-09-23
FI3430043T3 (fi) 2023-11-17
HRP20231464T1 (hr) 2024-03-01
CY1123784T1 (el) 2022-05-27
TWI784945B (zh) 2022-12-01
JOP20210085A1 (ar) 2023-01-30
MA43717B1 (fr) 2023-11-30
EP3219726B1 (de) 2020-12-02
PH12018501907A1 (en) 2019-06-17
US11459383B2 (en) 2022-10-04
TW202311288A (zh) 2023-03-16
JP2022130407A (ja) 2022-09-06
PL3219726T3 (pl) 2021-07-19
KR102470235B1 (ko) 2022-11-24
JP2019512268A (ja) 2019-05-16
HRP20210123T1 (hr) 2021-03-05
DK3430043T3 (da) 2023-11-27
AU2017235387B2 (en) 2024-06-27
UY37157A (es) 2017-10-31
CN109071650B (zh) 2022-05-13
NZ745999A (en) 2024-08-30
HUE052773T2 (hu) 2021-05-28
BR112018016667A2 (pt) 2019-02-05
CA3011784A1 (en) 2017-09-21
AR107917A1 (es) 2018-06-28
LT3219726T (lt) 2021-01-25
CR20180448A (es) 2019-01-25
SA518400027B1 (ar) 2022-03-06
EP4275745A3 (de) 2023-12-20
IL261031A (en) 2018-10-31
JOP20170064B1 (ar) 2021-08-17
ZA202108602B (en) 2022-03-30
AU2017235387A1 (en) 2018-09-27
KR20220158877A (ko) 2022-12-01
ZA202108599B (en) 2022-03-30
KR20180120149A (ko) 2018-11-05
ZA202108600B (en) 2022-03-30
CO2018009783A2 (es) 2018-09-28
AU2024219840A1 (en) 2024-10-10
RS64830B1 (sr) 2023-12-29
EP3430043A1 (de) 2019-01-23
SI3430043T1 (sl) 2024-01-31
CA3011784C (en) 2024-04-23
TWI829444B (zh) 2024-01-11
US12043663B2 (en) 2024-07-23
US20210198353A1 (en) 2021-07-01
TN2018000298A1 (en) 2020-01-16
ZA202108601B (en) 2022-03-30
MY194041A (en) 2022-11-09
EP3219726A1 (de) 2017-09-20
ES2836349T3 (es) 2021-06-24
US20230279091A1 (en) 2023-09-07
TW201739766A (zh) 2017-11-16
MA43717A (fr) 2018-11-28
EP4275745A2 (de) 2023-11-15
LT3430043T (lt) 2023-12-27
PT3430043T (pt) 2023-11-23
RS61374B1 (sr) 2021-02-26
MD3430043T2 (ro) 2024-02-29
US20230272059A1 (en) 2023-08-31
SG11201807062RA (en) 2018-09-27
ES2963221T3 (es) 2024-03-26
DK3219726T3 (da) 2020-12-07
GEP20217307B (en) 2021-10-25
EA201891788A1 (ru) 2019-01-31
CL2018002649A1 (es) 2018-11-09
SI3219726T1 (sl) 2021-02-26
HUE064238T2 (hu) 2024-02-28
MX2018011258A (es) 2019-02-13
EA039760B1 (ru) 2022-03-10
JP7129398B2 (ja) 2022-09-01
PT3219726T (pt) 2020-12-15

Similar Documents

Publication Publication Date Title
US12043663B2 (en) Anti-TNF-alpha-antibodies and functional fragments thereof
US10975143B2 (en) Anti-TNF-α-antibodies and functional fragments thereof
US10787508B2 (en) Anti-TNFα-antibodies and functional fragments thereof
US10759852B2 (en) Anti-TNF-alpha-antibodies and functional fragments thereof
AU2017235465B2 (en) Anti-TNFα-antibodies and functional fragments thereof

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 3011784

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 20187022632

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 261031

Country of ref document: IL

WWE Wipo information: entry into national phase

Ref document number: 122023003657

Country of ref document: BR

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112018016667

Country of ref document: BR

WWE Wipo information: entry into national phase

Ref document number: 11201807062R

Country of ref document: SG

WWE Wipo information: entry into national phase

Ref document number: 201891788

Country of ref document: EA

ENP Entry into the national phase

Ref document number: 14876

Country of ref document: GE

ENP Entry into the national phase

Ref document number: 2019500013

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: MX/A/2018/011258

Country of ref document: MX

Ref document number: CR2018-000448

Country of ref document: CR

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2017235387

Country of ref document: AU

Date of ref document: 20170316

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2017710748

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2017710748

Country of ref document: EP

Effective date: 20181017

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17710748

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 112018016667

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20180815

WWE Wipo information: entry into national phase

Ref document number: P-2023/1087

Country of ref document: RS